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Osteocytes and their cell processes reside in a large, interconnected network of voids pervading the mineralized bone matrix of most vertebrates. This osteocyte lacuno-canalicular network (OLCN) is believed to play important roles in mechanosensing, mineral homeostasis, and for the mechanical properties of bone. While the extracellular matrix structure of bone is extensively studied on ultrastructural and macroscopic scales, there is a lack of quantitative knowledge on how the cellular network is organized. Using a recently introduced imaging and quantification approach, we analyze the OLCN in different bone types from mouse and sheep that exhibit different degrees of structural organization not only of the cell network but also of the fibrous matrix deposited by the cells. We define a number of robust, quantitative measures that are derived from the theory of complex networks. These measures enable us to gain insights into how efficient the network is organized with regard to intercellular transport and communication. Our analysis shows that the cell network in regularly organized, slow-growing bone tissue from sheep is less connected, but more efficiently organized compared to irregular and fast-growing bone tissue from mice. On the level of statistical topological properties (edges per node, edge length and degree distribution), both network types are indistinguishable, highlighting that despite pronounced differences at the tissue level, the topological architecture of the osteocyte canalicular network at the subcellular level may be independent of species and bone type. Our results suggest a universal mechanism underlying the self-organization of individual cells into a large, interconnected network during bone formation and mineralization.

Significance A fundamental paradigm of bone biology is that the remodeling process—by which bones detect and repair damage—is orchestrated by osteocytes. The bones of most extant fish, however, lack these cells and should be unable to repair damage in their bones. We provide evidence for intense remodeling in the anosteocytic bone of billfishes, such as swordfish and marlin. Our observations challenge the central axiom that osteocytes alone are responsible for remodeling, suggesting alternate mechanisms in bone physiology and/or variation in the roles of bone cells. In addition, billfish bone exhibits an array of striking material properties that distinguish it from mammalian bone despite having similar composition, underlining that skeletal biology concepts are limiting when based on mammalian tissues alone. A remarkable property of tetrapod bone is its ability to detect and remodel areas where damage has accumulated through prolonged use. This process, believed vital to the long-term health of bone, is considered to be initiated and orchestrated by osteocytes, cells within the bone matrix. It is therefore surprising that most extant fishes (neoteleosts) lack osteocytes, suggesting their bones are not constantly repaired, although many species exhibit long lives and high activity levels, factors that should induce considerable fatigue damage with time. Here, we show evidence for active and intense remodeling occurring in the anosteocytic, elongated rostral bones of billfishes (e.g., swordfish, marlins). Despite lacking osteocytes, this tissue exhibits a striking resemblance to the mature bone of large mammals, bearing structural features (overlapping secondary osteons) indicating intensive tissue repair, particularly in areas where high loads are expected. Billfish osteons are an order of magnitude smaller in diameter than mammalian osteons, however, implying that the nature of damage in this bone may be different. Whereas billfish bone material is as stiff as mammalian bone (unlike the bone of other fishes), it is able to withstand much greater strains (relative deformations) before failing. Our data show that fish bone can exhibit far more complex structure and physiology than previously known, and is apparently capable of localized repair even without the osteocytes believed essential for this process. These findings challenge the unique and primary role of osteocytes in bone remodeling, a basic tenet of bone biology, raising the possibility of an alternative mechanism driving this process.

External ear canal (EEC) stenosis, often associated with cholesteatoma, carries a high risk of postoperative restenosis despite surgical intervention. While individualized implants offer promise in preventing restenosis, the high morphological variability of EECs and the lack of standardized definitions hinder systematic implant design. This study aimed to characterize individual EEC morphology and to develop a validated automated segmentation system for efficient implant preparation. Reference datasets were first generated by manual segmentation using 3D SlicerTM software version 5.2.2. Based on these, we developed a customized plugin capable of automatically identifying the maximal implantable region within the EEC and measuring its key dimensions. The accuracy of the plugin was assessed by comparing it with manual segmentation results in terms of shape, volume, length, and width. Validation was further performed using three temporal bone implantation experiments with 3D-Bioplotter©-fabricated EEC implants. The automated system demonstrated strong consistency with manual methods and significantly improved segmentation efficiency. The plugin-generated models enabled successful implant fabrication and placement in all validation tests. These results confirm the system’s clinical feasibility and support its use for individualized and systematic EEC implant design. The developed tool holds potential to improve surgical planning and reduce postoperative restenosis in EEC stenosis treatment.

The response of cells to changes in their physico-chemical micro-environment is essential to their survival. For example, bacterial magnetotaxis uses the Earth's magnetic field together with chemical sensing to help microorganisms move towards favoured habitats. The studies of such complex responses are lacking a method that permits the simultaneous mapping of the chemical environment and the response of the organisms, and the ability to generate a controlled physiological magnetic field. We have thus developed a multi-modal microscopy platform that fulfils these requirements. Using simultaneous fluorescence and high-speed imaging in conjunction with diffusion and aerotactic models, we characterized the magneto-aerotaxis of Magnetospirillum gryphiswaldense. We assessed the influence of the magnetic field (orientation; strength) on the formation and the dynamic of a micro-aerotactic band (size, dynamic, position). As previously described by models of magnetotaxis, the application of a magnetic field pointing towards the anoxic zone of an oxygen gradient results in an enhanced aerotaxis even down to Earth's magnetic field strength. We found that neither a ten-fold increase of the field strength nor a tilt of 45° resulted in a significant change of the aerotactic efficiency. However, when the field strength is zeroed or when the field angle is tilted to 90°, the magneto-aerotaxis efficiency is drastically reduced. The classical model of magneto-aerotaxis assumes a response proportional to the cosine of the angle difference between the directions of the oxygen gradient and that of the magnetic field. Our experimental evidence however shows that this behaviour is more complex than assumed in this model, thus opening up new avenues for research.

Modern therapy of inner ear disorders is increasingly shifting to local drug delivery using a growing number of pharmaceuticals. Access to the inner ear is usually made via the round window membrane (RWM), located in the bony round window niche (RWN). We hypothesize that the individual shape and size of the RWN have to be taken into account for safe reliable and controlled drug delivery. Therefore, we investigated the anatomy and its variations. Cone beam computed tomography (CBCT) images of 50 patients were analyzed. Based on the reconstructed 3D volumes, individual anatomies of the RWN, RWM, and bony overhang were determined by segmentation using 3D SlicerTM with a custom build plug-in. A large individual anatomical variability of the RWN with a mean volume of 4.54 mm3 (min 2.28 mm3, max 6.64 mm3) was measured. The area of the RWM ranged from 1.30 to 4.39 mm2 (mean: 2.93 mm2). The bony overhang had a mean length of 0.56 mm (min 0.04 mm, max 1.24 mm) and the shape was individually very different. Our data suggest that there is a potential for individually designed and additively manufactured RWN implants due to large differences in the volume and shape of the RWN.

The aim of this study was to develop and validate a semi-automated segmentation approach that identifies the round window niche (RWN) and round window membrane (RWM) for use in the development of patient individualized round window niche implants (RNI) to treat inner ear disorders. Twenty cone beam computed tomography (CBCT) datasets of unilateral temporal bones of patients were included in the study. Defined anatomical landmarks such as the RWM were used to develop a customized 3D Slicer™ plugin for semi-automated segmentation of the RWN. Two otolaryngologists (User 1 and User 2) segmented the datasets manually and semi-automatically using the developed software. Both methods were compared in-silico regarding the resulting RWM area and RWN volume. Finally, the developed software was validated ex-vivo in N = 3 body donor implantation tests with additively manufactured RNI. The independently segmented temporal bones of the different Users showed a strong consistency in the volume of the RWN and the area of the RWM. The volume of the semi-automated RWN segmentations were 48 ± 11% smaller on average than the manual segmentations and the area of the RWM of the semi-automated segmentations was 21 ± 17% smaller on average than the manual segmentation. All additively manufactured implants, based on the semi-automated segmentation method could be implanted successfully in a pressure-tight fit into the RWN. The implants based on the manual segmentations failed to fit into the RWN and this suggests that the larger manual segmentations were over-segmentations. This study presents a semi-automated approach for segmenting the RWN and RWM in temporal bone CBCT scans that is efficient, fast, accurate, and not dependent on trained users. In addition, the manual segmentation, often positioned as the gold-standard, actually failed to pass the implantation validation.

Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.

The formation of different tissues in the callus during secondary bone healing is at least partly influenced by mechanical stimuli. We use computer simulations to test the consequences of different hypotheses of the mechanoregulation at the cellular level on the patterns of tissues formed during healing. The computational study is based on an experiment on sheep, where after a tibial osteotomy, histological sections were harvested at different time points. In the simulations, we used a recently proposed basic phenomenological model, which allows ossification to occur either via endochondral or intramembranous ossification, but tries otherwise to employ a minimal number of simulation parameters. The model was extended to consider also the possibility of bone resorption and consequently allowing a description of the full healing progression till the restoration of the cortex. Specifically, we investigated how three changes in the mechanoregulation influence the resulting tissue patterns: (1) a time delay between stimulation of the cell and the formation of the tissue, (2) a variable mechanosensitivity of the cells, and (3) an independence of long time intervals of the soft tissue maturation from the mechanical stimulus. For all three scenarios, our simulations do not show qualitative differences in the time development of the tissue patterns. Largest differences were observed in the intermediate phases of healing in the amount and location of the cartilage. Interestingly, the course of healing was virtually unaltered in case of scenario (3) where tissue maturation proceeded independent of mechanical stimulation.

Osteocytes and their cell processes reside in a large, interconnected network of voids pervading the mineralized bone matrix of most vertebrates. This osteocyte lacuno-canalicular network (OLCN) is believed to play important roles in mechanosensing, mineral homeostasis, and for the mechanical properties of bone. While the extracellular matrix structure of bone is extensively studied on ultrastructural and macroscopic scales, there is a lack of quantitative knowledge on how the cellular network is organized. Using a recently introduced imaging and quantification approach, we analyze the OLCN in different bone types from mouse and sheep that exhibit different degrees of structural organization not only of the cell network but also of the fibrous matrix deposited by the cells. We define a number of robust, quantitative measures that are derived from the theory of complex networks. These measures enable us to gain insights into how efficient the network is organized with regard to intercellular transport and communication. Our analysis shows that the cell network in regularly organized, slow-growing bone tissue from sheep is less connected, but more efficiently organized compared to irregular and fast-growing bone tissue from mice. On the level of statistical topological properties (edges per node, edge length and degree distribution), both network types are indistinguishable, highlighting that despite pronounced differences at the tissue level, the topological architecture of the osteocyte canalicular network at the subcellular level may be independent of species and bone type. Our results suggest a universal mechanism underlying the self-organization of individual cells into a large, interconnected network during bone formation and mineralization.