μCT Measurements Research Articles

Gut Microbiome Manipulation Promotes Bone Anabolism via Regulatory T‐Cell Differentiation in Obese Mice

Probiotics are viable bacterial components that exert beneficial effects on the host, have been shown to prevent multiple comorbidities. Probiotics mostly act by changing the composition of the gut microbiota and increase gastrointestinal barrier integrity, which leads to the modulation of the immune system. However, the effects of probiotics in the pathological setting like obesity‐induced osteoporotic bone phenotype and their immunomodulatory effect are largely obscure. Therefore, we aimed to investigate the beneficial effects of probiotics accompanied by the high‐fat diet (HFD) on the bone loss and immune‐regulation at the molecular and epigenetic levels. To test this hypothesis, 10‐week‐old female mice were fed on the high‐fat diet (HFD; 42%) for 12 weeks. Probiotics (GSL#5) were given at the dose of 1 X 109 total bacteria 5 times/week through intragastric administration.ResultsIn vivo, μCT measurements revealed that HFD induced a significant increase of bone loss in trabecular bone phenotype (BV/TV; 42 %, BMD; 30% in the femur) and probiotics administration can able to prevent the bone loss. In vitro study revealed that probiotics induce mouse BMMSC derived osteoblast mineralization in HFD condition. In contrary, osteoclast differentiation and function were reduced as analyzed for the presence of RANK+, CD11b+ cells by flow cytometry. Mechanistic studies revealed that probiotics epigenetically regulates immune‐bone homeostasis by up‐regulating bone marrow (BM) regulatory T cells (Tregs CD4+Foxp3hi) differentiation, a suppressive population of naïve CD4+ T cells. Western blots and ChIP assays revealed that Tregs maintained expression of methylcytosine dioxygenases Tet1 and Tet2 via sulfhydration of nuclear transcription factor Y subunit beta (NFYB) to facilitate its binding to Tet1 and Tet2 promoters. Thereby Tet1 and Tet2 mediated catabolism of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC) in CpG islands of the Foxp3 promoter to establish a Tregs‐specific Foxp3 hypomethylation and Tregs differentiation. Further, Tregs induced the release of the IL10 and TGF‐β, leading to inhibit osteoclastogenesis and bone resorption, while increased bone formation under probiotics treatment. In vivo blockade of Tregs expansion by treatment with anti CD25 Ab or silencing of Foxp3 by siRNA based RNA interference in mice prevented the bone anabolic activity of probiotics. Collectively, these data provide the evidence that gut microbiota dysbiosis deregulates immune system; that is critical for osteoporotic bone loss in HFD induced obese female mice. Our data further suggest that the probiotic treatment in obesity‐induced osteoporotic bone loss seems to have a novel therapeutic potential via Treg/IL‐10 dependent pathway.Support or Funding InformationThis work is financially supported by National Institute of health grant AR‐067667 and HL‐107640‐NT are greatly acknowledged.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel mouse model of human prostate cancer to study intraprostatic tumor growth and the development of lymph node metastases

In this study, we aimed to establish a versatile in vivo model of prostate cancer, which adequately mimics intraprostatic tumor growth, and the natural routes of metastatic spread. In addition, we analyzed the capability of high-resolution ultrasonography (hrUS), in vivo micro-CT (μCT), and 9.4T MRI to monitor tumor growth and the development of lymph node metastases. A total of 5 × 105 VCaP cells or 5 × 105 cells of LuCaP136- or LuCaP147 spheroids were injected into the prostate of male CB17-SCID mice (n = 8 for each cell type). During 12 weeks of follow-up, orthotopic tumor growth, and metastatic spread were monitored by repetitive serum-PSA measurements and imaging studies including hrUS, μCT, and 9.4T MRI. At autopsy, primary tumors and metastases were harvested and examined by histology and immunohistochemistry (CK5, CK8, AMACR, AR, Ki67, ERG, and PSA). From imaging results and PSA-measurements, tumor volume doubling time, tumor-specific growth rate, and PSA-density were calculated. All 24 mice developed orthotopic tumors. The tumor growth could be reliably monitored by a combination of hrUS, μCT, MRI, and serum-PSA measurements. In most animals, lymph node metastases could be detected after 12 weeks, which could also be well visualized by hrUS, and MRI. Immunohistochemistry showed positive signals for CK8, AMACR, and AR in all xenograft types. CK5 was negative in VCaP- and focally positive in LuCaP136- and LuCaP147-xenografts. ERG was positive in VCaP- and negative in LuCaP136- and LuCaP147-xenografts. Tumor volume doubling times and tumor-specific growth rates were 21.2 days and 3.9 %/day for VCaP-, 27.6 days and 3.1 %/day for LuCaP136- and 16.2 days and 4.5 %/day for LuCaP147-xenografts, respectively. PSA-densities were 433.9 ng/mL per milliliter tumor for VCaP-, 6.5 ng/mL per milliliter tumor for LuCaP136-, and 11.2 ng/mL per milliliter tumor for LuCaP147-xenografts. By using different monolayer and 3D spheroid cell cultures in an orthotopic xenograft model, we established an innovative, versatile in vivo model of prostate cancer, which enables the study of both intraprostatic tumor growth as well as metastatic spread to regional lymph nodes. HrUS and MRI are feasible tools to monitor tumor growth and the development of lymph node metastases while these cannot be visualized by μCT.

3D visualized tracing of rebar corrosion-inhibiting features in concrete with a novel chemical self-healing system

A novel chemical self-healing system, with sodium monofluorophosphate/ethyl cellulose microcapsules, is designed with the objective of inhibiting rebar corrosion in concrete materials under NaCl solution. The corrosive behavior is traced and analyzed in 3D visualization by the method of micro X-ray computed microtomography (X-ray μCT). Environmental scanning electron microscopy (ESEM) and texture element analysis microscopy (TEAM) are used to verify the results from the X-ray μCT measurement. The experimental results show that typical pitting corrosion features are detected directly by X-ray μCT. The chemical self-healing system, with sodium monofluorophosphate/ethyl cellulose microcapsules, can postpone the rust procedure and effectively inhibit the rust rate of rebar in a simulated corrosion environment. The corrosion-inhibiting performance becomes significant with the microcapsules increasing. Moreover, a linear relationship between volume losses of rebar and time is deduced from the X-ray μCT data. Corresponding corrosion ratios and corrosion rates are quantitatively calculated for comparison of the inhibition afforded by the chemical self-healing system.

Effect of the geometrical defectiveness on the mechanical properties of SLM biomedical Ti6Al4V lattices

Metallic lattice biomaterials can be very complex structures that are often impossible to be fabricated with other manufacturing technologies than additive manufacturing (AM). Residual stresses and geometric defects such as severe notches and distorted struts are inevitably introduced into the printed structures and these can affect the mechanical and biological properties. Micro X-ray Computed Tomography (µCT) has been proven to be a very powerful tool for accurately measuring the mismatch between the as-designed CAD model and the SLM structure. In this work, selective laser melting (SLM) Ti6Al4V lattices were measured using a metrological µCT system to identify and classify the geometrical distortions introduced by the printing process. The µCT measurements have also been used to build Finite Element (FE) models based on beam elements that make possible a quantification of the effect of these defects on the elastic modulus of the lattice by comparison with FE models based on the ideal geometry. Moreover, solid FE models of the junctions between the struts have been built by importing the CT data in Ansys® to calculate the stress concentrations caused by the severe notches.

Galectin-3 as a novel regulator of osteoblast-osteoclast interaction and bone homeostasis

Bone tissue undergoes permanent and lifelong remodeling with a concerted action of bone-building osteoblasts and bone-resorbing osteoclasts. A precise cooperation between those two cell types is critical in the complex process of bone renewal. Galectin-3 is a member of the β-galactoside-binding lectin family playing multiple roles in cell growth, differentiation and aggregation. As it has been described to be expressed in bone, galectin-3 might influence bone homeostasis by regulating the function and/or interplay of osteoblasts and osteoclasts. Here, we investigated the role of galectin-3 in osteoclastogenesis and osteoblast-osteoclast interactions. Bone histomorphometric analysis and μCT measurements revealed a decreased trabecular bone volume and an increased osteoclast number in 12weeks old male galectin-3 knockout mice compared to wildtype littermates. Galectin-3 deficient bone marrow cells displayed a higher osteoclastogenic capacity in ex vivo differentiation assays, associated with elevated TRAF6 mRNA levels, suggesting an intrinsic inhibition of osteoclastogenesis by galectin-3 interfering with RANKL-mediated signaling. Furthermore, the addition of extracellular galectin-3 to murine or human osteoclastogenesis assays inhibited osteoclast formation and osteoclast numbers were higher in co-culture assays with galectin-3 deficient osteoblasts. In conclusion, our data suggest the secretion of galectin-3 as a novel mechanism for osteoblasts to control osteoclastogenesis and to maintain trabecular bone homeostasis independently of the RANKL/OPG-axis.

The Nanoporous RP-20 Carbon Electrode as a Model for Energy Storage and Conversion Systems – Studied with µCT, SAXS and SANS Techniques

The manuscript describes quantitative morphological and structural information for the nanoporous RP-20 powder andelectrode based on that carbon - studied with X-ray microcomputed tomography, small-angle neutron scattering, and small-angle X-ray scattering techniques. μCT measurements demonstrated that the diffusion process inside of the RP-20 electrode is significantly influenced by the nature of the solvent. SAXS measurements were performed in order to collect quantitative morphological and structural information for nanoporous RP-20 powder-polytetrafluoroethylene binder based electrode material. Comparison of the complementary scattering techniques, i.e., SAXS and SANS has been provided. The interaction of heavy water inside of the nanoporous PR-20 based electrode by SANS technique was observed.

Porous piezoelectric ceramics with 3-3-connectivity fabricated by impregnation of cellulose paper structures

Porous piezoelectric ceramics with an interconnecting pore network (3-3 piezoceramic-air-composites) are a compromising material for sensor applications. Using a biomorphous approach with pre-pyrolysed cellulose fiber paper as template for the impregnation process leads to a novel porous piezoelectric ceramic with a fiber microstructure and benefits on easy shaping of complex structures and high porosity (∼90vol%). The templates were impregnated by a non-aqueous lead zirconate titanate (PZT) slurry (solid fracture of 52.3%). After residual slurry and binder removal and cellulose/carbon burn-out, sintering was carried out at 1150°C. Microstructure and composition analyses were done by µCT (“Skyscan 1172”, Bruker), SEM (FESEM, Helios NanoLab 600i FIB Workstation, FEI) and X-ray diffraction measurements (Kristalloflex D500, Siemens AG) evaluation. Geometric density decreased by 30% compared to dense PZT (as reference) a porosity increased from 2.4% up to 86%. The piezoelectric properties as d33, relative permittivity and d31 (measured by “Piezo-Meter PM 300″, Piezo Test) decreased up to 99%.

Effect of reinforcement size and orientation on the thermal expansion behavior of metallic glass reinforced metal matrix composites produced by gas pressure infiltration

Many efforts were made during the last years to utilize the superior properties of metallic glasses as reinforcements in metal matrix composites and several investigations were carried out to characterize composites’ mechanical properties. Since there is a complete lack of knowledge about their thermal properties, this work focuses on the thermal expansion behavior of metallic glass reinforced metal matrix composites produced via gas pressure infiltration. The aluminum alloy AlSi12 was used as matrix material. Metallic glass Ni60Nb20Ta20 flakes with different size ranges were taken as reinforcement and volume fractions in the range of 29–44% were studied. X-ray micro computed tomography (μCT) measurements were performed to investigate the reinforcement 3D-structure within the composite. The thermal expansion behavior depending on flake orientation within the composite was investigated by dilatometric measurements. Four thermal cycles were carried out between room temperature and 500°C at a constant heating and cooling rate of 5°C/min. Results show that thermal strain rather depends on flake size and orientation than on volume fraction. Further, the composites exhibit a distinct anisotropic behavior for the thermal expansion coefficient (CTE) due to a layered flake structure within the composite. Finally determined CTEs were compared with several thermo-mechanical models to study the underlying mechanisms of the composite behavior.

The Nanoporous RP-20 Carbon Electrode As a Model for Energy Storage and Conversion Systems – Studied with µ CT, SAXS and SANS Methods

There is no doubt that the electrochemical energy storage and conversion devices are the key components in future’s smart energy grids enabling on-demand and efficient energy usage. During the last thirty years, various carbon based materials have been studied and used in the energy storage and conversion devices: supercapacitors, fuel cells and batteries. Despite the efforts made so far, there is still a strong need for better understanding of processes related to the mass transport properties of electrolyte ions inside of the porous electrode materials driven by their morphology [1,2]. It is obvious that electrochemical performance (durability/efficiency) of such a device depends on the rate of the charging and discharging processes inside the electrodes that in turn are strongly influenced by the properties of the carbon material and electrolyte used (i.e., the specific surface area, pore shape, size/volume, wettability, ionophilicity/ionophobicity, dielectrical permittivity and polarizability, ect.) [3,4]. It has been recognized that different carbon materials exhibit different properties such as the surface area, hierarchical porosity, electrical and ionic conductivity, water uptake rate as well as the electrocatalytic activity [5,6]. The scientific goal of this study is in depth understanding of the fundamental properties of the carbon materials and their functionality at the molecular and atomic level under non-polarized conditions. For this purpose the nanoporous carbon RP-20 [7] as a main electrode component has been used and correlation between morphology (i.e., pore shape, size of the pores) and instance wetting (filling density) of the carbon material matrix with the solvent was determined. Keeping in mind real application requirements, the primarily used 1M NaClO4 solution in dimethyl carbonate (DMC) and ethylene carbonate (EC) (1:1 wt%) solvent mixture as an electrolyte [3] has been studied. Several independent methods – x-ray microcomputed tomography (µCT) [8], small-angle neutron scattering (SANS) [9], and small-angle x-ray scattering (SAXS) [10-12] were applied for characterization of the following systems: RP-20 1MNaClO4 in EC:DMC; RP-20 in EC:DMC or RP-20 1MNaClO4 in D2O. It is rather difficult to simultaneously determine/distinguish the structure or dynamic effects on the efficiency and durability of the electrochemical devices (i.e., inhomogeniously/randomly distributed pores filled with electrolyte contributing during the charge and discharge cycling processes), therefore the above listed methods have contributed to an improved awareness of the nature of complex systems in steady state (non-polarized) conditions. Based on the µCT measurements, the linear attenuation coefficient values and filling densities were obtained and calculated. To determine the shape and size of the pores for nanoporous RP-20 electrode and to observe the influence/interaction of the electrolyte ions dissolved in aqueous and non-aqueous solvent (i.e., differences in filling of the pores) SANS method was applied. SAXS measurements were performed in order to collect quantitative morphological and structural information for nanoporous RP-20 powder and RP-20 electrode material (e.g., surface area, pore size, pore structure, binder effect).

MiR-375-3p negatively regulates osteogenesis by targeting and decreasing the expression levels of LRP5 and β-catenin.

Wnt signaling pathways are essential for bone formation. Previous studies showed that Wnt signaling pathways were regulated by miR-375. Thus, we aim to explore whether miR-375 could affect osteogenesis. In the present study, we investigated the roles of miR-375 and its downstream targets. Firstly, we revealed that miR-375-3p negatively modulated osteogenesis by suppressing positive regulators of osteogenesis and promoting negative regulators of osteogenesis. In addition, the results of TUNEL cell apoptosis assay showed that miR-375-3p induced MC3T3-E1 cell apoptosis. Secondly, miR-375-3p targeted low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor of the Wnt signaling pathways, and β-catenin as determined by luciferase activity assay, and it decreased the expression levels of LRP5 and β-catenin. Thirdly, the decline of protein levels of β-catenin was determined by immunocytochemistry and immunofluorescence. Finally, silence of LRP5 in osteoblast precursor cells resulted in diminished cell viability and cell proliferation as detected by WST-1-based colorimetric assay. Additionally, all the parameters including the relative bone volume from μCT measurement suggested that LRP5 knockout in mice resulted in a looser and worse-connected trabeculae. The mRNA levels of important negative modulators relating to osteogenesis increased after the functions of LRP5 were blocked in mice. Last but not least, the expression levels of LRP5 increased during the osteogenesis of MC3T3-E1, while the levels of β-catenin decreased in bone tissues from osteoporotic patients with vertebral compression fractures. In conclusion, we revealed miR-375-3p negatively regulated osteogenesis by targeting LRP5 and β-catenin. In addition, loss of functions of LRP5 damaged bone formation in vivo. Clinically, miR-375-3p and its targets might be used as diagnostic biomarkers for osteoporosis and might be also as novel therapeutic agents in osteoporosis treatment. The relevant products of miR-375-3p might be developed into molecular drugs in the future. These molecules could be used in translational medicine.

Mitogen-inducible gene-6 partly mediates the inhibitory effects of prenatal dexamethasone exposure on endochondral ossification in long bones of fetal rats.

Prenatal exposure to dexamethasone slows down fetal linear growth and bone mineralization but the regulatory mechanism remains unknown. Here we assessed how dexamethasone regulates bone development in the fetus. Dexamethasone (1mg·kg(-1) ·day(-1) ) was injected subcutaneously every morning in pregnant rats from gestational day (GD)9 to GD20. Fetal femurs and tibias were harvested at GD20 for histological and gene expression analysis. Femurs of 12-week-old female offspring were harvested for microCT (μCT) measurement. Primary chondrocytes were treated with dexamethasone (10, 50, 250 and 1000nM). Prenatal dexamethasone exposure resulted in accumulation of hypertrophic chondrocytes and delayed formation of the primary ossification centre in fetal long bone. The retardation was accompanied by reduced maturation of hypertrophic chondrocytes, decreased osteoclast number and down-regulated expression of osteocalcin and bone sialoprotein in long bone. In addition, the mitogen-inducible gene-6 (Mig6) and osteoprotegerin (OPG) expression were stimulated, and the receptor activator of NF-κB ligand (RANKL) expression was repressed. Moreover, dexamethasone activated OPG and repressed RANKL expression in both primary chondrocytes and primary osteoblasts, and the knockdown of Mig6 abolished the effect of dexamethasone on OPG expression. Further, μCT measurement showed loss of bone mass in femur of 12-week-old offspring with prenatal dexamethasone exposure. Prenatal dexamethasone exposure delays endochondral ossification by suppressing chondrocyte maturation and osteoclast differentiation, which may be partly mediated by Mig6 activation in bone. Bone development retardation in the fetus may be associated with reduced bone mass in later life.

High-resolution peripheral quantitative computed tomography (HR-pQCT) can assess microstructural and biomechanical properties of both human distal radius and tibia: Ex vivo computational and experimental validations

High-resolution peripheral quantitative computed tomography (HR-pQCT) provides in vivo three-dimensional (3D) imaging at the distal radius and tibia and has been increasingly used to characterize cortical and trabecular bone morphology in clinical studies. In this study, we comprehensively examined the accuracy of HR-pQCT and HR-pQCT based micro finite element (μFE) analysis predicted bone elastic stiffness and strength through comparisons with gold-standard micro computed tomography (μCT) based morphological/μFE measures and direct mechanical testing results. Twenty-six sets of human cadaveric distal radius and tibia segments were imaged by HR-pQCT and μCT. Microstructural analyses were performed for the registered HR-pQCT and μCT images. Bone stiffness and yield strength were determined by both HR-pQCT and μCT based linear and nonlinear μFE predictions and mechanical testing. Our results suggested that strong and significant correlations existed between the HR-pQCT standard, model-independent and corresponding μCT measurements. HR-pQCT based nonlinear μFE overestimated stiffness and yield strength while the linear μFE underestimated yield strength, but both were strongly correlated with those predicted by μCT μFE and measured by mechanical testing at both radius and tibia (R2>0.9). The microstructural differences between HR-pQCT and μCT were also examined by the Bland–Altman plots. Our results showed HR-pQCT morphological measurements of BV/TVd, Tb.Th, and Tb.Sp, can be adjusted by correction values to approach true values measured by gold-standard μCT. In addition, we observed moderate correlations of HR-pQCT biomechanical and microstructural parameters between the distal radius and tibia. We concluded that morphological and mechanical properties of human radius and tibia bone can be assessed by HR-pQCT based measures.

Artificial extracellular matrix - a promising approach to enhance new bone formation in critical size bone defects

Event Abstract Back to Event Artificial extracellular matrix - a promising approach to enhance new bone formation in critical size bone defects Yvonne Förster1, Vera Hintze2, Stefanie Möller3, Matthias Schnabelrauch3, Dieter Scharnweber2 and Stefan Rammelt1 1 University Hospital "Carl Gustav Carus" at TU Dresden, UniversityCenter of Orthopedics and Trauma Surgery, Germany 2 TU Dresden, Institute of Materials Science, Max Bergmann Center of Biomaterials, Germany 3 Innovent e.V. Jena, Department Biomaterials, Germany Bone regeneration in critical size bone defects still represents an important but unsolved clinical problem. Still, autologous bone transplantation is the medical gold standard which is associated with donor site morbidity and limited resources. Biodegradable scaffolds are a promising alternative for bone regeneration as they provide porous matrices that can support cell adhesion, proliferation, and differentiation. The addition of specific coatings can improve the osteoconductive and osteoinductive properties of these scaffolds. Previous studies have shown the successful use of components of the organic extracellular matrix (ECM) like collagen type I (Coll) and glycosaminoglycans (GAGs) in order to create a favorable microenvironment for bone forming cells and their progenitors. An artificial ECM (aECM) consisting of Coll and chondroitin sulfate (CS) or sulfated hyaluronan (sHA) improved osteogenic differentiation of mesenchymal stromal cells in vitro[1],[2]. The aim of the present study was to investigate the effect of these a ECM on new bone formation in a critical size bone defect in vivo. A 5 mm defect was created in the femur of 84 adult male Wistar rats and stabilized with an internal fixator and randomly divided into 7 groups of 12 animals each. Embroidered PCL (polycaprolactone-co-lactide) scaffolds were coated with Coll, Coll/CS, Coll/hyaluronan (Coll/HA), Coll/oversulfated CS (Coll/sCS3, degree of sulfation (DS) ≈ 3), and Coll/sHA3 (DS ≈ 3). Uncoated PCL scaffolds served as negative controls, and collagen sponges with bone chips to mimic autologous bone graft served as positive controls. Six rats of each group were sacrificed after 2 and 12 weeks. New bone formation and the quality of the newly formed bone were evaluated by radiographs, micro computed tomography (µCT), and histological staining. Histological staining showed no signs of inflammation indicating a good biocompatibility of the coatings. Radiographs and µCT measurements showed nearly no formation of calcified tissue within the scaffolds at 2 weeks after implantation. HE staining at 2 weeks showed incipient ossification from the bone ends with small clusters of newly formed woven bone for Coll/CS- and Coll/sHA-coated scaffolds indicating an earlier bone remodeling compared to the controls. The new bone volume after 12 weeks was 77%±20% for the positive control, 51%±19% for the uncoated PCL, 46%±30% for Coll, 45%±27% for Coll/CS, 47%±30% for Coll/HA, 57%±24% for Coll/sCS3, and 73%±38% for Coll/sHA3. Thus, scaffolds coated with Coll/sHA3 displayed the same amount of new bone formation as the positive control. In this study we could demonstrate that the PCL scaffolds coated with an artificial ECM consisting of Coll and highly sulfated HA have the same effect on new bone formation as autologous bone grafting in a critical size bone defect in rats. In a clinical setting such scaffolds could potentially spare the patients an additional surgery for bone harvesting. Fig. 1: A) µCT images of selected coatings to illustrate the distribution of the new bone within the defect area at 12 weeks after implantation. B) Quantification of the new bone formation after 2 and 12 weeks of implantation. The contralateral section of the femur was used as reference and set to 100%. Data are represented as mean±SD. This study was supported by grants from Deutsche Forschungsgemeinschaft Transregio 67 (subprojects A3, B5, and Z3). The authors thank Heike Zimmermann, Aline Katzschner, Suzanne Manthey, and Annett Wenke for technical assistance.

Micro-CT in the Assessment of Pediatric Renal Osteodystrophy by Bone Histomorphometry.

Computed tomography (CT) measurements can distinguish between cortical and trabecular bone density in vivo. High-resolution CTs assess both bone volume and density in the same compartment, thus potentially yielding information regarding bone mineralization as well. The relationship between bone histomorphometric parameters of skeletal mineralization and bone density from microcomputed tomography (μCT) measurements of bone cores from patients on dialysis has not been assessed. Bone cores from 68 patients with ESRD (age =13.9±0.5 years old; 50% men) and 14 controls (age =15.3±3.8 years old; 50% men) obtained as part of research protocols between 1983 and 2006 were analyzed by bone histomorphometry and μCT. Bone histomorphometric diagnoses in the patients were normal to high bone turnover in 76%, adynamic bone in 13%, and osteomalacia in 11%. Bone formation rate did not correlate with any μCT determinations. Bone volume measurements were highly correlated between bone histomorphometry and μCT (bone volume/tissue volume between the two techniques: r=0.70; P<0.001, trabecular thickness and trabecular separation: r=0.71; P<0.001, and r=0.56; P<0.001, respectively). Osteoid accumulation as determined by bone histomorphometry correlated inversely with bone mineral density as assessed by μCT (osteoid thickness: r=-0.32; P=0.01 and osteoid volume: r=-0.28; P=0.05). By multivariable analysis, the combination of bone mineral density and bone volume (as assessed by μCT) along with parathyroid hormone and calcium levels accounted for 38% of the variability in osteoid volume (by histomorphometry). Measures of bone volume can be accurately assessed with μCT. Bone mineral density is lower in patients with excessive osteoid accumulation and higher in patients with adynamic, well mineralized bone. Thus, bone mineralization may be accurately assessed by μCT of bone biopsy cores. Additional studies are warranted to define the value of high-resolution CT in the prediction of bone mineralization in vivo.

Evaluation of in-vivo measurement errors associated with micro-computed tomography scans by means of the bone surface distance approach

In vivo micro-computed tomography (µCT) scanning is an important tool for longitudinal monitoring of the bone adaptation process in animal models. However, the errors associated with the usage of in vivo µCT measurements for the evaluation of bone adaptations remain unclear. The aim of this study was to evaluate the measurement errors using the bone surface distance approach. The right tibiae of eight 14-week-old C57BL/6 J female mice were consecutively scanned four times in an in vivo µCT scanner using a nominal isotropic image voxel size (10.4 µm) and the tibiae were repositioned between each scan. The repeated scan image datasets were aligned to the corresponding baseline (first) scan image dataset using rigid registration and a region of interest was selected in the proximal tibia metaphysis for analysis. The bone surface distances between the repeated and the baseline scan datasets were evaluated. It was found that the average (±standard deviation) median and 95th percentile bone surface distances were 3.10 ± 0.76 µm and 9.58 ± 1.70 µm, respectively. This study indicated that there were inevitable errors associated with the in vivo µCT measurements of bone microarchitecture and these errors should be taken into account for a better interpretation of bone adaptations measured with in vivo µCT.

Early changes in morphology, bone mineral density and matrix composition of vertebrae lead to disc degeneration in aged collagen IX −/− mice

Collagen IX (Col IX) is an important component of the cartilage extracellular matrix and has been associated with degenerative cartilage disorders and chondrodysplasias in humans. Further, polymorphisms in Col IX are known risk factors for the development of early intervertebral disc (IVD) degeneration. To understand the role of Col IX in the pathogenesis of IVD disorders, the spine of newborn and older Col IX deficient mice was systematically analyzed and compared to C57BL/6N controls. Morphology and bone parameters of the spine from newborn, 6 and 10months old animals were investigated using μCT measurements. Histological staining was used to evaluate tissue structure and degree of degeneration. Localization and expression of extracellular matrix proteins was analyzed in depth by immunofluorescence staining, immunoblotting, RT-PCR and in situ hybridization. High resolution imaging and stiffness measurements were performed by atomic force microscopy (AFM). Vertebral bodies of newborn Col IX-deficient mice were smaller and showed an increased mineral density compared to wild type animals. At birth, lack of Col IX led to a disrupted cellular organization in the cartilaginous endplate and a smaller nucleus pulposus of the IVD. Expression levels and localization of other extracellular matrix proteins were strongly altered accompanied by a softening of cartilaginous tissues. In older animals, absence of Col IX caused earlier and more pronounced disc degeneration with annular fissures.The absence of Col IX induces early developmental, structural and biomechanical alterations in both vertebral body and intervertebral disc which eventually cause severe degenerative changes in the aging spine.

The effect of pedicle screw implantation depth and angle on the loading and stiffness of a spinal fusion assembly.

The increasing prevalence of spine disorders in industrialized environments has impaired the quality of life in the elder population. In an effort to relieve pain, physicians strive to improve treatment through the consideration of patient specific characteristics during preoperative planning of procedures such as spinal fusion. This study aims at quantifying aspects of spondylodesis to the loading and mobility of the utilized instrumentation, as the use of rigid vs. motion sparing materials as well as implantation angle and depth of the pedicle screws are still subject to controversy among surgeons. A fixation assembly was reverse engineered based on µCT measurements of the involved instrumentation. Two pedicle screws were connected with a rod, thus representing a mono-segmental fixation device. The pedicle screws were embedded in hexahedral structures simulated by bone properties. Upon validation and verification, the response of the model to a compressive and a torsional load was simulated in ANSYS 14, while altering the implantation depth and insertion angle of the pedicle screws along with the rod material. The mobility of the instrumentation was drastically increased (by up to 390%) when PEEK rods were used in place of traditional Ti ones, a tendency observed at varying extent for all simulated scenarios. Shallow implantation induced a slight stress increase (∼21%) on the implant and a notable distressing of the bony tissue (∼44%), whereas inclined screw positioning was overall beneficial to the developing stress fields in both, with bone profiting a max. stress release of ∼15% during the application of torsion. The investigation presented refined insight into the biomechanical response of a spinal fusion device. As expected, rigid fixation seems preferable in fusion oriented instrumentation whereas semi rigid devices should be employed for non-fusion applications. Shallow implantation resulted in a slight posterior offset of the stabilization device, which could be beneficial in the treatment of osteoporotic patients.

0349 : Comparison of bioimpedance and X-ray micro-computed tomography (μCT) for total fat volume measurement in mice in vivo

Determination of adipose tissue proportion, as well as its spatial distribution, is a major part of metabolic syndrome assessment. Various methods, commonly used in mouse models, are either indirect measurement, invasive (destructive) or imaging techniques. Indirect measurement techniques, such as deuterium dilution (considered as gold standard) or bioimpedance are based on the measurement of body composition. They give access to the measure of total body fat, without distinction between visceral and subcutaneous fat volumes. Sacrifice and ex-vivo tissue weight allow both total fat measurement and partition between subcutaneous and visceral fat pad. It is a direct method that does not allow for longitudinal studies. Non-invasive micro-imaging modalities (magnetic resonance imaging and μCT) add, over others, the advantage of spatial localization of fat, in vivo. Of our study was to compare results of measurements of total body fat done by μCT and bioimpedance in mice. μCT was done using a dedicated in vivo imaging instrument (eXplore Vision 120, GE, USA) on 30 anesthetized mice. Each acquisition lasted approximately 12 min. Just after μCT, measurement of body composition was performed by bioimpedance (ImpediVET, ImpediMed, USA). The average value of total body fat by μCT is 5% higher than that obtained by bioimpedance. The standard deviation calculated from μCT measurements is lower than that obtained with bioimpedance (4.8% vs 6.8% respectively). The linear correlation of μCT and bioimpedance measure of total adipose tissue is moderate (r 2 = 0.55). It is the first time, to our knowledge, that a comparison of total fat volume measurement between μCT and bioimpedance is performed in mice. Surprisingly, the correlation appears not to be so trivial between the 2 methods. Before a deeper exploration, comparison of results obtained with these 2 methods should be made with caution.

Homogenization of elastic properties of short-fiber reinforced composites based on measured microstructure data

Mechanical properties of short-fiber reinforced composites are crucially influenced by their microstructure. The microstructure itself is mainly governed by the manufacturing process like injection or compression molding. The main contribution of this paper lies in the homogenization of linear elastic properties using experimental microstructural information. For this purpose, the microstructure of injection-molded specimens made of polypropylene reinforced with 30wt.% of short glass fibers are analyzed through micro-computer tomography ( μCT) measurements. Applying a recently developed segmentation algorithm, the spatial position, the orientation distribution and the length of the fibers are determined. This data is evaluated in terms of orientation tensors and length distribution, and is used within three mean field approaches: a self-consistent homogenization method, the interaction direct derivative estimate, which is based on the three-phase model, and a two-step bounding method. All methods account for the orientation, the length and the diameter distribution. The numerical results are compared to experimental tensile tests.

Pore-scale analysis of formation damage in Bentheimer sandstone with in-situ NMR and micro-computed tomography experiments

Abstract We investigated fines movement through sandstone in-situ at the micrometre pore scale and studied the associated pore-scale mechanisms leading to formation damage. We used two in-situ techniques to accomplish this, namely nuclear magnetic resonance T 2 relaxation time (NMR) measurements (of pore size distributions) and high resolution x-ray micro-computed tomography (μCT; at high resolutions of (0.89 μm) 3 and (3.4 μm) 3 ). The μCT images showed the precise 3D location of the fines particles in the plug and demonstrated that initially pore throats are plugged, followed by filling of adjacent pore bodies by solid particles. These measurements in combination with traditionally used (indirect) permeability and production curve measurements and ex-situ SEM imaging enabled us to propose a new mechanistic pore-scale plugging model; furthermore we demonstrated that the amount of fines trapped decayed rapidly with core depth. We conclude that it is feasible to analyse formation damage in-situ by a combination of NMR and μCT measurements.