Gene expression detection in developing mouse tissue using in situ hybridization and µCT imaging.

High resolution and noninvasiveness have made soft-tissue X-ray microtomography (µCT) a widely applicable three-dimensional (3D) imaging method in studies of morphology and development. However, scarcity of molecular probes to visualize gene activity with µCT has remained a challenge. Here, we apply horseradish peroxidase-assisted reduction of silver and catalytic gold enhancement of the silver deposit to in situ hybridization in order to detect gene expression in developing tissues with µCT (here called GECT, gene expression CT). We show that GECT detects expression patterns of collagen type II alpha 1 and sonic hedgehog in developing mouse tissues comparably with an alkaline phosphatase-based detection method. After detection, expression patterns are visualized with laboratory µCT, demonstrating that GECT is compatible with varying levels of gene expression and varying sizes of expression regions. Additionally, we show that the method is compatible with prior phosphotungstic acid staining, a conventional contrast staining approach in µCT imaging of soft tissues. Overall, GECT is a method that can be integrated with existing laboratory routines to obtain spatially accurate 3D detection of gene expression.

Assessment of post-trauma microstructural alterations in the rabbit knee cartilage and subchondral bone.

Early diagnosis of post-traumatic osteoarthritis (PTOA) is critical for designing better treatments before the degradation becomes irreversible. We utilized multimodal high-resolution imaging to investigate early-stage deterioration in articular cartilage and the subchondral bone plate from a sub-critical impact to the knee joint, which initiates PTOA. The knee joints of 12 adult rabbits were mechanically impacted once on the femoral articular surface to initiate deterioration. At 2- and 14-week post-impact surgery, cartilage-bone blocks were harvested from the impact region in the animals (N = 6 each). These blocks were assessed for deterioration using polarized light microscopy (PLM), microcomputed tomography (µCT), and biochemical analysis. Statistically significant changes were noted in the impact tissues across the calcified zone (CZ) at 14 weeks post-impact: the optical retardation values in the CZ of impact cartilage had a drop of 29.0% at 14 weeks, while the calcium concentration in the CZ of impact cartilage also had a significant drop at 14 weeks. A significant reduction of 6.3% in bone mineral density (BMD) was noted in the subchondral bone plate of the impact samples at 14 weeks. At 2 weeks post-impact, only minor, non-significant changes were measured. Furthermore, the impact knees after 14 weeks had greater structural changes compared with the 2-week impact knees, indicating progressive degradation over time. The findings of this study facilitated a connection between mineralization alterations and the early deterioration of knee cartilage after a mechanical injury. In a broader context, these findings can be beneficial in improving clinical strategies to manage joint injuries.

Pre-operative bone quality deficits and risk of complications following spine fusion surgery among postmenopausal women.

Poor bone quality is a risk factor for complications after spinal fusion surgery. This study investigated pre-operative bone quality in postmenopausal women undergoing spine fusion and found that those with small bones, thinner cortices and surgeries involving more vertebral levels were at highest risk for complications. PURPOSE: Spinal fusion is one of the most common surgeries performed worldwide. While skeletal complications are common, underlying skeletal deficits are often missed by pre-operative DXA due to artifact from spinal pathology. This prospective cohort study investigated pre-operative bone quality using high resolution peripheral CT (HRpQCT) and its relation to post-operative outcomes in postmenopausal women, a population that may be at particular risk for skeletal complications. We hypothesized that women with low volumetric BMD (vBMD) and abnormal microarchitecture would have higher rates of post-operative complications. METHODS: Pre-operative imaging included areal BMD (aBMD) by DXA, cortical and trabecular vBMD and microarchitecture of the radius and tibia by high resolution peripheral CT. Intra-operative bone quality was subjectively graded based on resistance to pedicle screw insertion. Post-operative complications were assessed by radiographs and CTs. RESULTS: Among 50 women enrolled (age 65 years), mean spine aBMD was normal and 35% had osteoporosis by DXA at any site. Low aBMD and vBMD were associated with "poor" subjective intra-operative quality. Skeletal complications occurred in 46% over a median follow-up of 15 months. In Cox proportional models, complications were associated with greater number of surgical levels (HR 1.19 95% CI 1.06-1.34), smaller tibia total area (HR 1.67 95% CI1.16-2.44) and lower tibial cortical thickness (HR 1.35 95% CI 1.05-1.75; model p < 0.01). CONCLUSION: Women with smaller bones, thinner cortices and procedures involving a greater number of vertebrae were at highest risk for post-operative complications, providing insights into surgical and skeletal risk factors for complications in this population.

Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

Iron overload is one of the secondary osteoporosis etiologies. Cellular and molecular mechanisms involved in iron-related osteoporosis are not fully understood. AIM: The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. MATERIAL AND METHODS: We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. RESULTS: Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 µM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. CONCLUSION: Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

A novel simplified approach for endodontic retrograde surgery in short single-rooted teeth.

BACKGROUND: High technical thresholds, long operative times, and the need for expensive and specialized equipment impede the widespread adoption of endodontic microsurgery in many developing countries. This study aimed to compare the effects of a simplified, cost-effective, and time-efficient surgical approach involving orthograde obturation using biological ceramic material greater than 6 mm combined with apicoectomy for single-rooted teeth with short lengths with those of the conventional and current standard methods. MATERIALS AND METHODS: Forty-five premolars equally categorized into three groups: conventional surgery group, standard surgery group, and modified surgery group. A µCT scan was used to calculate the volume of voids. A micro-leakage test and scanning electron microscope (SEM) were performed to assess the sealing effect. Additionally, four cases of chronic periapical periodontitis in the anterior region were selected, and the patients received either the modified approach or the standard surgery for endodontic microsurgery. RESULTS: The volumes of voids in the apical 0-3 mm of the modified group and the standard group were comparable. The micro-leakage test and SEM examination demonstrated closely bonded fillings in the dentinal walls in both the modified surgery group and standard surgery group. The outcomes of the preliminary application of this modified procedure on patients were successful at the time of the follow-up cutoff. CONCLUSIONS: The modified surgery group exhibited similar root canal filling and apical sealing abilities with the standard procedure for single-rooted teeth with short lengths (< 20 mm). The preliminary application of this modified surgical procedure achieved favorable results.

The visible skeleton 2.0: phenotyping of cartilage and bone in fixed vertebrate embryos and foetuses based on X-ray microCT.

For decades, clearing and staining with Alcian Blue and Alizarin Red has been the gold standard to image vertebrate skeletal development. Here, we present an alternate approach to visualise bone and cartilage based on X-ray microCT imaging, which allows the collection of genuine 3D data of the entire developing skeleton at micron resolution. Our novel protocol is based on ethanol fixation and staining with Ruthenium Red, and efficiently contrasts cartilage matrix, as demonstrated in whole E16.5 mouse foetuses and limbs of E14 chicken embryos. Bone mineral is well preserved during staining, thus the entire embryonic skeleton can be imaged at high contrast. Differences in X-ray attenuation of ruthenium and calcium enable the spectral separation of cartilage matrix and bone by dual energy microCT (microDECT). Clearing of specimens is not required. The protocol is simple and reproducible. We demonstrate that cartilage contrast in E16.5 mouse foetuses is adequate for fast visual phenotyping. Morphometric skeletal parameters are easily extracted. We consider the presented workflow to be a powerful and versatile extension to the toolkit currently available for qualitative and quantitative phenotyping of vertebrate skeletal development.

Spatial organization and connectivity of wood rays in Pinus massoniana xylem based on high-resolution µCT-assisted network analysis.

MAIN CONCLUSION: Spatial organization and connectivity of wood rays in Pinus massoniana was comprehensively viewed and regarded as anatomical adaptions to ensure the properties of rays in xylem. Spatial organization and connectivity of wood rays are essential for understanding the wood hierarchical architecture, but the spatial information is ambiguous due to small cell size. Herein, 3D visualization of rays in Pinus massoniana was performed using high-resolution µCT. We found brick-shaped rays were 6.5% in volume fractions, nearly twice the area fractions estimated by 2D levels. Uniseriate rays became taller and wider during the transition from earlywood to latewood, which was mainly contributed from the height increment of ray tracheids and widened ray parenchyma cells. Furthermore, both volume and surface area of ray parenchyma cells were larger than ray tracheids, so ray parenchyma took a higher proportion in rays. Moreover, three different types of pits for connectivity were segmented and revealed. Pits in both axial tracheids and ray tracheids were bordered, but the pit volume and pit aperture of earlywood axial tracheids were almost tenfold and over fourfold larger than ray tracheids. Contrarily, cross-field pits between ray parenchyma and axial tracheids were window-like with the principal axis of 31.0 µm, but its pit volume was approximately one-third of axial tracheids. Additionally, spatial organization of rays and axial resin canal was analyzed by a curved surface reformation tool, providing the first evidence of rays close to epithelial cells inward through the resin canal. Epithelial cells had various morphologies and large variations in cell size. Our results give new insights into the organization of radial system of xylem, especially the connectivity of rays with adjacent cells.

3D reconstruction of the bronchial tree of the Gray short-tailed opossum (Monodelphis domestica) in the postnatal period.

Recent didelphid marsupials resemble the assumed mammalian ancestor and are suitable to inform on the evolution of the mammalian lung. This study uses X-ray computed tomography (µCT) to three-dimensionally reconstruct the bronchial tree of the marsupial Gray short-tailed opossum (Monodelphis domestica) in order to reveal the timeline of morphogenesis during the postnatal period. The development of the bronchial tree was examined in 37 animals from embryonic day 13, during the postnatal period (neonate to 57 days) and in adults. The first appearance and the branching of lobar, segmental and sub-segmental bronchioles in the lungs were documented. Based on the reconstructions, the generation of end-branching airways, the median and maximum generation and the number of branches were calculated for each pulmonary lobe. At birth, the lung of M. domestica has a primitive appearance since it consists of a simple system of branching airways that end in a number of terminal air spaces, lobar bronchioles, and first segmental bronchioles are present. During the postnatal period, the volumes of the lung and bronchial tree steadily increase and development, differentiation, and expansion of the bronchial tree takes place. By 14 days, the fundamental bronchial tree consisting of lobar, segmental, and sub-segmental bronchioles has been established. A mature bronchial tree, including respiratory bronchioles and alveolar ducts is present by day 35. The asymmetry of the right (predominately four lobes) and the left lung (predominately two lobes), as present in M. domestica, can be considered as plesiomorphic for Mammalia. In marsupials, the process of branching morphogenesis, which takes place intrauterine in the placental fetus, is shifted to the postnatal period, but follows similar patterns as described in placentals. Lung maturation in general and the branching morphogenesis in particular seems to be highly conservative within mammalian evolution.

Descriptive catalog of Homo naledi dental remains from the 2013 to 2015 excavations of the Dinaledi Chamber, site U.W. 101, within the Rising Star cave system, South Africa.

More than 150 hominin teeth, dated to ∼330-241 thousand years ago, were recovered during the 2013-2015 excavations of the Dinaledi Chamber of the Rising Star cave system, South Africa. These fossils comprise the first large single-site sample of hominin teeth from the Middle Pleistocene of Africa. Though scattered remains attributable to Homo sapiens, or their possible lineal ancestors, are known from older and younger sites across the continent, the distinctive morphological feature set of the Dinaledi teeth supports the recognition of a novel hominin species, Homo naledi. This material provides evidence of African Homo lineage diversity that lasts until at least the Middle Pleistocene. Here, a catalog, anatomical descriptions, and details of preservation and taphonomic alteration are provided for the Dinaledi teeth. Where possible, provisional associations among teeth are also proposed. To facilitate future research, we also provide access to a catalog of surface files of the Rising Star jaws and teeth.

The mechanoresponse of bone is closely related to the osteocyte lacunocanalicular network architecture.

Organisms rely on mechanosensing mechanisms to adapt to changes in their mechanical environment. Fluid-filled network structures not only ensure efficient transport but can also be employed for mechanosensation. The lacunocanalicular network (LCN) is a fluid-filled network structure, which pervades our bones and accommodates a cell network of osteocytes. For the mechanism of mechanosensation, it was hypothesized that load-induced fluid flow results in forces that can be sensed by the cells. We use a controlled in vivo loading experiment on murine tibiae to test this hypothesis, whereby the mechanoresponse was quantified experimentally by in vivo micro-computed tomography (µCT) in terms of formed and resorbed bone volume. By imaging the LCN using confocal microscopy in bone volumes covering the entire cross-section of mouse tibiae and by calculating the fluid flow in the three-dimensional (3D) network, we could perform a direct comparison between predictions based on fluid flow velocity and the experimentally measured mechanoresponse. While local strain distributions estimated by finite-element analysis incorrectly predicts preferred bone formation on the periosteal surface, we demonstrate that additional consideration of the LCN architecture not only corrects this erroneous bias in the prediction but also explains observed differences in the mechanosensitivity between the three investigated mice. We also identified the presence of vascular channels as an important mechanism to locally reduce fluid flow. Flow velocities increased for a convergent network structure where all of the flow is channeled into fewer canaliculi. We conclude that, besides mechanical loading, LCN architecture should be considered as a key determinant of bone adaptation.

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy.

An improved understanding of an ovary's structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin's solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues.

A versatile laboratory setup for high resolution X-ray phase contrast tomography and scintillator characterization.

BACKGROUND: X-ray micro-tomography (µCT) is a powerful non-destructive 3D imaging method applied in many scientific fields. In combination with propagation-based phase-contrast, the method is suitable for samples with low absorption contrast. Phase contrast tomography has become available in the lab with the ongoing development of micro-focused tube sources, but it requires sensitive and high-resolution X-ray detectors. The development of novel scintillation detectors, particularly for microscopy, requires more flexibility than available in commercial tomography systems. OBJECTIVE: We aim to develop a compact, flexible, and versatile µCT laboratory setup that combines absorption and phase contrast imaging as well as the option to use it for scintillator characterization. Here, we present details on the design and implementation of the setup. METHODS: We used the setup for µCT in absorption and propagation-based phase-contrast mode, as well as to study a perovskite scintillator. RESULTS: We show the 2D and 3D performance in absorption and phase contrast mode, as well as how the setup can be used for testing new scintillator materials in a realistic imaging environment. A spatial resolution of around 1.3µm is measured in 2D and 3D. CONCLUSIONS: The setup meets the needs for common absorption µCT applications and offers increased contrast in phase contrast mode. The availability of a versatile laboratory µCT setup allows not only for easy access to tomographic measurements, but also enables a prompt monitoring and feedback beneficial for advances in scintillator fabrication.

Complexity untwined: The structure and function of cucumber (Cucumis sativus L.) shoot phloem.

Cucurbit phloem is complex, with large sieve tubes on both sides of the xylem (bicollateral phloem), and extrafascicular elements that form an intricate web linking the rest of the vasculature. Little is known of the physical interconnections between these networks or their functional specialization, largely because the extrafascicular phloem strands branch and turn at irregular angles. Here, export in the phloem from specific regions of the lamina of cucumber (Cucumis sativus L.) was mapped using carboxyfluorescein and 14 C as mobile tracers. We also mapped vascular architecture by conventional microscopy and X-ray computed tomography using optimized whole-tissue staining procedures. Differential gene expression in the internal (IP) and external phloem (EP) was analyzed by laser-capture microdissection followed by RNA-sequencing. The vascular bundles of the lamina form a nexus at the petiole junction, emerging in a predictable pattern, each bundle conducting photoassimilate from a specific region of the blade. The vascular bundles of the stem interconnect at the node, facilitating lateral transport around the stem. Elements of the extrafascicular phloem traverse the stem and petiole obliquely, joining the IP and EP of adjacent bundles. Using pairwise comparisons and weighted gene coexpression network analysis, we found differences in gene expression patterns between the petiole and stem and between IP and EP, and we identified hub genes of tissue-specific modules. Genes related to transport were expressed primarily in the EP while those involved in cell differentiation and development as well as amino acid transport and metabolism were expressed mainly in the IP.

In situ, direct observation of seasonal embolism dynamics in Aleppo pine trees growing on the dry edge of their distribution.

Xylem embolism impairs hydraulic conductivity in trees and drives drought-induced mortality. While embolism has been monitored in vivo in potted plants, and research has revealed evidence of embolism in field-grown trees, continuous in situ monitoring of cavitation in forests is lacking. Seasonal patterns of embolism were monitored in branchlets of Aleppo pine (Pinus halepensis) trees growing in a dry Mediterranean forest. Optical visualization (OV) sensors were installed on terminal branches, in addition to monthly sampling for micro computed tomography scans. We detected 208 cavitation events among four trees, which represented an embolism increase from zero to c. 12% along the dry season. Virtually all the cavitation events occurred during daytime hours, with 77% occurring between 10:00 and 17:00 h. The probability for cavitation in a given hour increased as vapor pressure deficit (VPD) increased, up to a probability of 42% for cavitation when VPD > 5 kPa. The findings uniquely reveal the instantaneous environmental conditions that lead to cavitation. The increased likelihood of cavitation events under high VPD in water-stressed pines is the first empirical support for this long hypothesized relationship. Our observations suggest that low levels of embolism are common in Aleppo pine trees at the dry edge of their distribution.

Inactivation of Osteoblast PKC Signaling Reduces Cortical Bone Mass and Density and Aggravates Renal Osteodystrophy in Mice with Chronic Kidney Disease on High Phosphate Diet.

Chronic kidney disease (CKD) frequently leads to hyperphosphatemia and hyperparathyroidism, mineral bone disorder (CKD-MBD), ectopic calcifications and cardiovascular mortality. PTH activates the osteoanabolic Gαs/PKA and the Gαq/11/PKC pathways in osteoblasts, the specific impact of the latter in CKD-MBD is unknown. We generated osteoblast specific Gαq/11 knockout (KO) mice and established CKD-MBD by subtotal nephrectomy and dietary phosphate load. Bone morphology was assessed by micro-CT, osteoblast function by bone planar scintigraphy at week 10 and 22 and by histomorphometry. Osteoblasts isolated from Gαq/11 KO mice increased cAMP but not IP3 in response to PTH 1-34, demonstrating the specific KO of the PKC signaling pathway. Osteoblast specific Gαq/11 KO mice exhibited increased serum calcium and reduced bone cortical thickness and mineral density at 24 weeks. CKD Gαq/11 KO mice had similar bone morphology compared to WT, while CKD Gαq/11-KO on high phosphate diet developed decreased metaphyseal and diaphyseal cortical thickness and area, as well as a reduction in trabecular number. Gαq/11-KO increased bone scintigraphic tracer uptake at week 10 and mitigated tracer uptake in CKD mice at week 22. Histological bone parameters indicated similar trends. Gαq/11-KO in osteoblast modulates calcium homeostasis, bone formation rate, bone morphometry, and bone mineral density. In CKD and high dietary phosphate intake, osteoblast Gαq/11/PKC KO further aggravates mineral bone disease.

The Role of Type VI Collagen in Alveolar Bone.

Many studies have been conducted to elucidate the role of Type VI collagen in muscle and tendon, however, its role in oral tissues remains unclear. In this study, an α2(VI) deficient mouse (Col6α2-KO) model was used to examine the role of Type VI collagen in oral tissues. Tissue volume and mineral density were measured in oral tissues by µCT. Proteome analysis was performed using protein extracted from alveolar bone. In addition, alveolar bone was evaluated with a periodontitis induced model. µCT analysis showed the Col6α2-KO mice had less volume of alveolar bone, dentin and dental pulp, while the width of periodontal ligament (PDL) was greater than WT. The mineral density in alveolar bone and dentin were elevated in Col6α2-KO mice compared with WT. Our proteome analysis showed significant changes in proteins related to ECM organization and elevation of proteins associated with biomineralization in the Col6α2-KO mice. In induced periodontitis, Col6α2-KO mice had greater alveolar bone loss compared with WT. In conclusion, Type VI collagen has a role in controlling biomineralization in alveolar bone and that changes in the ECM of alveolar bone could be associated with greater bone loss due to periodontitis.

Evaluation of the Efficacy of Two Fibre-Reinforced Post Removal Techniques.

OBJECTIVE: The aim of this in-vitro study was to compare the amount of material (post and luting agent) and root dentine removed using two methods for removal of endodontic posts. METHODS: Human premolar teeth (n=20) were sectioned at the CEJ and fibre reinforced posts were cemented at a length of 10mm following root canal therapy. Teeth were randomly assigned to two study groups. The methods of removal compared were: the use of RTD re-access kit (Composipost, RTD, France, St Egreve) in a conventional hand-piece driven by an electric motor (Group A); or a long tapered diamond bur (FG Diamond grit bur, Dentsply Ltd, UK) in an air-driven high speed turbine (Group B). Using micro- CT the volume of material and root dentine removed for each sample was calculated. RESULTS: Both the volume of material removed and the volume of root dentine removed were significantly greater when using diamond burs. (p⟨0.001). The volume of dentine removed using the diamond bur method (mean 22.64mm³) was greater than the volume removed using the reaccess kit (mean 11.71mm³). SIGNIFICANCE: Use of a diamond bur to remove fibre reinforced endodontic post removal poses higher risk for root perforation compared to the reaccess kit.

Exploring the macrostructural anatomy of dendrochirotid sea cucumber's (Echinodermata) calcareous rings under micro-computed tomography and its bearing on phylogeny.

Despite descending from heavily calcified ancestors, the holothuroid skeleton is fully internal and composed of microscopic ossicles and a ring of plates bound by connective tissue, the calcareous ring. The calcareous ring exhibits a complex and poorly understood morphology; as a result, establishing unambiguous homology statements about its macrostructure has been challenging and phylogenetic studies have had to simplify this important structure. Here, we provide the first broad comparative study of Dendrochirotida calcareous rings using micro-computed tomography (µCT). A detailed description of the three-dimensional macrostructure of the calcareous ring of 10 sea cucumber species, including rare and type specimens, is presented. The structures observed were highly variable at the subfamily level, especially at the point of tissue attachment. The relationship between the calcareous ring and its associated organs, and their functional morphology are discussed. To aid future phylogenetic studies, we listed 22 characters and performed a preliminary cladistic analysis. The topology obtained supports the idea that the simple, cucumariid ring is ancestral to the mosaic-like phyllophorid ring; however, it did not support the monophyly of the cucumariids. It also did not support the family Sclerodactylidae, which was described based on the ring morphology. Differently from the dermal ossicles, which are highly homoplastic, the general homoplasy index of the calcareous ring characters was relatively low. This result highlights the importance of this structure for phylogenetic inference. Unfortunately, time since collection, rough collection methods and fixation can damage the skeleton, and the calcareous ring is often overlooked in taxonomic descriptions. The data presented here will improve our understanding of holothuroid relationships and facilitate studies on holothuroid functional morphology and biomechanics.

Morphology and serum and bone tissue calcium and magnesium concentrations in the bones of male rats chronically treated with letrozole, a nonsteroidal cytochrome P450 aromatase inhibitor.

Purpose/Aim of the study: The role of estrogen (E) in the regulation of bone turnover in women is well established, though the contributions of E versus testosterone (T) in the control of bone turnover in men are poorly understood. The aim of this study was to examine the association between chronic treatment with letrozole, a nonsteroidal inhibitor blocking the aromatase activity and thus the conversion of androgens into estrogens, and cortical bone morphology in the femur and humerus of male adult rats.Materials and Methods: Adult male rats were treated with letrozole for 6 months and the body and femur weight, morphology, collagen structure, blood serum, and bone tissue concentrations of calcium and magnesium were examined.Results: Long-term aromatase inhibition resulted in a decrease in femur mass, a wavelike arrangement of bone and lamellae with an altered organization of collagen in compact bone, a increased concentration of calcium in blood serum, and no change in calcium bone tissue concentration, magnesium serum, or bone tissue concentration. MicroCT study of the humerus revealed significant decreases of whole bone tissue volume, cortical bone thickness, cortical bone volume, and external cortical bone thickness with letrozole treatment.Conclusion: Chronic treatment with letrozole affected cortical bone structure and produced histomorphological changes in male rat bone similar to that observed in the aging processes.

Automated resolution independent method for comparing in vivo and dry trabecular bone.

OBJECTIVES: Variation in human trabecular bone morphology can be linked to habitual behavior, but it is difficult to investigate in vivo due to the radiation required at high resolution. Consequently, functional interpretations of trabecular morphology remain inferential. Here we introduce a method to link low- and high-resolution CT data from dry and fresh bone, enabling bone functional adaptation to be studied in vivo and results compared to the fossil and archaeological record. MATERIALS AND METHODS: We examine 51 human dry bone distal tibiae from Nile Valley and UK and two pig tibiae containing soft tissues. We compare low-resolution peripheral quantitative computed tomography (pQCT) parameters and high-resolution micro CT (µCT) in homologous single slices at 4% bone length and compare results to our novel Bone Ratio Predictor (BRP) method. RESULTS: Regression slopes between linear attenuation coefficients of low-resolution pQCT images and bone area/total area (BA/TA) of high-resolution µCT scans differ substantially between geographical subsamples, presumably due to diagenesis. BRP accurately predicts BA/TA (R2 = .97) and eliminates the geographic clustering. BRP accurately estimates BA/TA in pigs containing soft tissues (R2 = 0.98) without requiring knowledge of true density or phantom calibration of the scans. DISCUSSION: BRP allows automated comparison of image data from different image modalities (pQCT, µCT) using different energy settings, in archeological bone and wet specimens. The method enables low-resolution data generated in vivo to be compared with the fossil and archaeological record. Such experimental approaches would substantially improve behavioral inferences based on trabecular bone microstructure.