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Osteocytes are mechanosensitive, bone-embedded cells which are connected via dendrites in a lacuno-canalicular network and regulate bone resorption and formation balance. Alterations in osteocyte lacunar volume, shape and density have been identified in conditions of aging, osteoporosis and osteolytic bone metastasis, indicating patterns of impaired bone remodeling, osteolysis and disease progression. Osteolytic bone disease is a hallmark of the hematologic malignancy multiple myeloma (MM), in which monoclonal plasma cells in the bone marrow disrupt the bone homeostasis and induce excessive resorption at local and distant sites. Qualitative and quantitative changes in the 3D osteocyte lacunar morphometry have not yet been evaluated in MM, nor in the precursor conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). In this study, we characterized the osteocyte lacunar morphology in trabecular bone of the iliac crest at the ultrastructural level using high resolution microCT in human bone biopsy samples of three MGUS, two SMM and six newly diagnosed MM. In MGUS, SMM and MM we found a trend for lower lacunar density and a shift towards larger lacunae with disease progression (higher 50 % cutoff of the lacunar volume cumulative distribution) in the small osteocyte lacunae 20-900 µm3 range compared to control samples. In the larger lacunae 900-3000 µm3 range, we detected significantly higher lacunar density and microporosity in the MM group compared to the MGUS/SMM group. Regarding the shape distribution, the MGUS/SMM group showed a trend for flatter, more elongated and anisotropic osteocyte lacunae compared to the control group. Altogether, our findings suggest that osteocytes in human MM bone disease undergo changes in their lacunae density, volume and shape, which could be an indicator for osteolysis and disease progression. Future studies are needed to understand whether alterations of the lacunae architecture affect the mechanoresponsiveness of osteocytes, and ultimately bone adaptation and fracture resistance in MM and its precursors conditions.

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In vitro testing for evaluating degradation mode and rate of candidate biodegradable metals to be used as intravascular stents is crucial before going to in vivo animal models. In this study, we show that X-ray microfocus computed tomography (microCT) presents a key added value to visualize degradation mode and to evaluate degradation rate and material surface properties in 3D and at high resolution of large regions of interest. The in vitro degradation behavior of three candidate biodegradable stent materials was evaluated: pure iron (Fe), pure zinc (Zn), and a quinary Zn alloy (ZnAgCuMnZr). These metals were compared to a reference biostable cobaltchromium (CoCr) alloy. To compare the degradation mode and degradation rate evaluated with microCT, scanning electron microscopy (SEM) and inductively-coupled plasma (ICP) were included. We confirmed that Fe degrades very slowly but with desirable uniform surface corrosion. Zn degrades faster but exhibits localized deep pitting corrosion. The Zn alloy degrades at a similar rate as the pure Zn, but more homogeneously. However, the formation of deep internal dendrites was observed. Our study provides a detailed microCT-based comparison of essential surface and corrosion properties, with a structural characterization of the corrosion behavior, of different candidate stent materials in 3D in a non-destructive way.

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The aim of this study was to probe an unexpected relationship between the ice nucleation temperature (TIN), process efficiency and product attributes in a controlled ice nucleation (CIN) lyophilization process. An amorphous product was lyophilized with (CIN-5 °C, CIN-7 °C or CIN-10 °C) or without (NOCIN) control of ice nucleation. Process parameters and product attributes were monitored and compared using a series of advanced in-line and off-line process analytical technology (PAT) tools. Unexpectedly, an indirect relationship was observed between TIN and primary drying efficiency for the CIN processes. Further, the CIN-5 °C process was associated with higher product resistance to mass flow than corresponding CIN-7 °C and CIN-10 °C processes. Surprisingly, the air voids in some NOCIN products were larger than CIN-5 °C products but comparable to CIN-7 °C. Heat flux analysis revealed an indirect relationship between TIN and the minimum hold time required to complete solidification. The heat flux analysis also revealed all products underwent complete solidification prior to primary drying. The order of homogeneity in water activity of the products was CIN-5 °C ≥NOCIN>CIN-7 °C. The higher homogeneity in water activity of CIN-5 °C than corresponding CIN-7 °C processes indicated that the lower process efficiency of CIN-5 °C could not be attributed to unsuccessful induction of ice nucleation during CIN-5 °C. High resolution micro-CT imaging and Artificial Intelligence Image analysis revealed cake wall deformation in CIN-7 °C and NOCIN products but not in CIN-5 °C. In addition, NOCIN products had bimodal distribution in air voids with median size range of 4-5 µm and 151.9-309 µm, respectively, hence the lower process efficiency of NOCIN despite the higher D90. Thus, the observed relationship between TIN and process efficiency may be attributed to microstructural changes post freezing. This hypothesis was corroborated by visible macroscopic cake collapse in NOCIN products but not in CIN products after lyophilization at a higher shelf temperature. In conclusion, the advantages of controlling the ice nucleation temperature of a lyophilization process may only be attained through a robust process design that takes into consideration the primary and secondary drying process parameters. Further, combined use of advanced in-line and off-line PAT tools for process and product characterization may hasten the at scale adoption of advance techniques such as CIN.

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High-resolution non-destructive 3D microCT imaging allows the visualization and structural characterization of mineralized cartilage and bone. Deriving statistically relevant quantitative structural information about these tissues, however, requires automated segmentation procedures, mainly because manual contouring is user-biased and time-consuming. Despite the increased spatial resolution in microCT 3D volumes, automatic segmentation of mineralized cartilage versus bone remains non-trivial since they have similar grayscale values. Our work investigates how reliable 2D segmentation masks can be predicted automatically based on a (set of) convolutional neural network(s) trained with a limited number of manually annotated samples. To do that, we compared different strategies to select the 2D samples to annotate and considered ensemble learning and test-time augmentation (TTA) to mitigate the limited accuracy and robustness resulting from the small number of annotated training samples. We show that, for a fixed amount of annotated image samples, 2D microCT slices to annotate should preferably be selected in distinct 3D volumes, at regular intervals, rather than being grouped in adjacent slices of a same 3D volume. Two main lessons are drawn regarding the use of ensembles or TTA instead of a single model. First, ensemble learning is shown to improve segmentation accuracy and to reduce the mean and standard deviation of the absolute errors in cartilage characteristics obtained with different initializations of the neural network training process. In contrast, TTA appears to be unable to improve the model's robustness to unlucky initializations. Second, both TTA and ensembling improved the model's confidence in its predictions and segmentation failure detection.

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Skeletal adaptation is substantially influenced by mechanical loads. Osteocytes and their lacuno-canalicular network have been identified as a key player in load sensation and bone quality regulation. In the femoral neck, one of the most common fracture sites, a complex loading pattern with lower habitual loading in the superolateral neck and higher compressive stresses in the inferomedial neck is present. Variations in the femoral neck-shaft angle (NSA), i.e., coxa vara or coxa valga, provide the opportunity to examine the influence of loading patterns on bone quality. We obtained femoral neck specimens of 28 osteoarthritic human subjects with coxa vara, coxa norma and coxa valga during total hip arthroplasty. Bone mineral density (BMD) was assessed preoperatively by dual energy X-ray absorptiometry (DXA). Cortical and trabecular microstructure and three-dimensional osteocyte lacunar characteristics were assessed in the superolateral and inferomedial neck using ex vivo high resolution micro-computed tomography. Additionally, BMD distribution and osteocyte lacunar characteristics were analyzed by quantitative backscattered electron imaging (qBEI). All groups presented thicker inferomedial than superolateral cortices. Furthermore, the superolateral site exhibited a lower osteocyte lacunar density along with lower lacunar sphericity than the inferomedial site, independent of NSA. Importantly, BMD and corresponding T-scores correlated with microstructural parameters at the inferomedial but not superolateral neck. In conclusion, we provide micromorphological evidence for fracture vulnerability of the superolateral neck, which is independent of NSA and BMD. The presented bone qualitative data provide an explanation why DXA may be insufficient to predict a substantial proportion of femoral neck fractures. STATEMENT OF SIGNIFICANCE: The femoral neck, one of the most common fracture sites, is subject to a complex loading pattern. Site-specific differences (i.e., superolateral vs. inferomedial) in bone quality influence fracture risk, but it is unclear how this relates to hip geometry and bone mineral density (BMD) measurements in vivo. Here, we examine femoral neck specimens using a variety of high-resolution imaging techniques and demonstrate impaired bone quality in the superolateral compared to the inferomedial neck. Specifically, we found impaired cortical and trabecular microarchitecture, mineralization, and osteocyte properties, regardless of neck-shaft angle. Since BMD correlated with bone quality of the inferomedial but not the superolateral neck, our results illustrate why bone densitometry may not predict a substantial proportion of femoral neck fractures.

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Three-dimensional reconstruction plays a vital role in assisting doctors and surgeons in diagnosing the healing progress of bone defects. Common three-dimensional reconstruction methods include surface and volume rendering. As the focus is on the shape of the bone, this study omits the volume rendering methods. Many improvements have been made to surface rendering methods like Marching Cubes and Marching Tetrahedra, but not many on working towards real-time or near real-time surface rendering for large medical images and studying the effects of different parameter settings for the improvements. Hence, this study attempts near real-time surface rendering for large medical images. Different parameter values are experimented on to study their effect on reconstruction accuracy, reconstruction and rendering time, and the number of vertices and faces. The proposed improvement involving three-dimensional data smoothing with convolution kernel Gaussian size 5 and mesh simplification reduction factor of 0.1 is the best parameter value combination for achieving a good balance between high reconstruction accuracy, low total execution time, and a low number of vertices and faces. It has successfully increased reconstruction accuracy by 0.0235%, decreased the total execution time by 69.81%, and decreased the number of vertices and faces by 86.57% and 86.61%, respectively.

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OBJECTIVE: To characterize the prenatal and postnatal craniofacial bone development in mouse model of all-trans retinoic acid (ATRA) exposure at different ages by a quantitative and morphological analysis of skull morphology. METHODS: Pregnant mice were exposed to ATRA at embryonic day 10 (E10) and 13 (E13) by oral gavage. Skulls of mice embryos at E19.5 and adult mice at postnatal day 35 (P35) were collected for high-resolution microcomputed tomography (microCT) imaging scanning and section HE staining. Reconstruction and measurement of mouse skulls were performed for prenatal and postnatal analysis of the control and ATRA-exposed mice. RESULTS: Craniofacial malformations in mouse models caused by ATRA exposure were age dependent. ATRA exposure at E10 induced cleft palate in 81.8% of the fetuses, whereas the palatine bone of E13-exposed mice was intact. Inhibitions of maxilla and mandible development with craniofacial asymmetry induced were observed at E19.5 and P35. Compared with control and E13-exposed mice, the palatine bones of E10-exposed mice were not elevated and were smaller in dimension. Some E10-exposed mice exhibited other craniofacial abnormalities, including premature fusion of mandibular symphysis with a missing mandibular incisor and a smaller mandible. Severe deviated snouts and amorphous craniofacial suture were detected in E13-exposed mice at P35. CONCLUSION: These morphological variations in E10- and E13-exposed mice suggested that ATRA was teratogenic in craniofacial bone development in mice and the effect was age dependent.

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Measurements of perilymph hydrodynamics in the human cochlea are scarce, being mostly limited to the fluid pressure at the basal or apical turn of the scalae vestibuli and tympani. Indeed, measurements of fluid pressure or volumetric flow rate have only been reported in animal models. In this study we imaged the human ear at 6.7 and 3-µm resolution using µCT scanning to produce highly accurate 3D models of the entire ear and particularly the cochlea scalae. We used a contrast agent to better distinguish soft from hard tissues, including the auditory canal, tympanic membrane, malleus, incus, stapes, ligaments, oval and round window, scalae vestibule and tympani. Using a Computational Fluid Dynamics (CFD) approach and this anatomically correct 3D model of the human cochlea, we examined the pressure and perilymph flow velocity as a function of location, time and frequency within the auditory range. Perimeter, surface, hydraulic diameter, Womersley and Reynolds numbers were computed every 45° of rotation around the central axis of the cochlear spiral. CFD results showed both spatial and temporal pressure gradients along the cochlea. Small Reynolds number and large Womersley values indicate that the perilymph fluid flow at auditory frequencies is laminar and its velocity profile is plug-like. The pressure was found 102-106° out of phase with the fluid flow velocity at the scalae vestibule and tympani, respectively. The average flow velocity was found in the sub-µm/s to nm/s range at 20-100Hz, and below the nm/s range at 1-20kHz.

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Frogs (Lissamphibia: Anura) use adhesive tongues to capture fast moving, elusive prey. For this, the tongues are moved quickly and adhere instantaneously to various prey surfaces. Recently, the functional morphology of frog tongues was discussed in context of their adhesive performance. It was suggested that the interaction between the tongue surface and the mucus coating is important for generating strong pull-off forces. However, despite the general notions about its importance for a successful contact with the prey, little is known about the surface structure of frog tongues. Previous studies focused almost exclusively on species within the Ranidae and Bufonidae, neglecting the wide diversity of frogs. Here we examined the tongue surface in nine different frog species, comprising eight different taxa, i.e., the Alytidae, Bombinatoridae, Megophryidae, Hylidae, Ceratophryidae, Ranidae, Bufonidae, and Dendrobatidae. In all species examined herein, we found fungiform and filiform papillae on the tongue surface. Further, we observed a high degree of variation among tongues in different frogs. These differences can be seen in the size and shape of the papillae, in the fine-structures on the papillae, as well as in the three-dimensional organization of subsurface tissues. Notably, the fine-structures on the filiform papillae in frogs comprise hair-like protrusions (Megophryidae and Ranidae), microridges (Bufonidae and Dendrobatidae), or can be irregularly shaped or absent as observed in the remaining taxa examined herein. Some of this variation might be related to different degrees of adhesive performance and may point to differences in the spectra of prey items between frog taxa.

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3D highly porous (93% total porosity) gelatin scaffolds were prepared according to a novel, simple method, which implies gelatin foaming, gelification, soaking into ethanol and successive freeze-drying. Reinforcement of the as-prepared scaffolds (GEL) was performed through immersion in aqueous solutions at different gelatin concentrations. Reinforcement solutions with and without genipin addition allowed to prepare two series of samples:cross-linked and uncross-linked samples, respectively. The amount of gelatin adsorbed onto the reinforced samples increases as a function of gelatin concentration in solution and provokes a drastic improvement of the compressive modulus and collapse strength up to values of about 30 and 4 MPa, respectively. The open and interconnected porosity, although slightly reduced, is still of the order of 80% in the samples reinforced with the highest concentration of gelatin. Water uptake ability evaluated after immersion in PBS for 20 s decreases with gelatin reinforcement. The presence of genipin in cross-linked samples reduces gelatin release and stabilizes the scaffolds in solution. Chondrocytes from human articular cartilage adhere, proliferate, and penetrate into the scaffolds. The evaluation of differentiation markers both on the supernatants of cell culture and by means of quantitative polymerase chain reaction (qPCR) indicates a dose-dependent promotion of cell differentiation.

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