Evan M. Johnson

In Vivo Respiratory-Gated Micro-CT Imaging in Small-Animal Oncology Models

Micro-computed tomography(micro-CT) is becoming an accepted research tool for the noninvasive examination of laboratory animals such as mice and rats, but to date, in vivo scanning has largely been limited to the evaluation of skeletal tissues. We use a commercially available micro-CT device to perform respiratory gated in vivo acquisitions suitable for thoracic imaging. The instrument is described, along with the scan protocol and animal preparation techniques. Preliminary results confirm that lung tumors as small as 1 mm in diameter are visible in vivo with these methods. Radiation dose was evaluated using several approaches, and was found to be approximately 0.15 Gy for this respiratory-gated micro-CT imaging protocol. The combination of high-resolution CT imaging and respiratory-gated acquisitions appears well-suited to serial in vivo scanning.

Stiffness of the endplate boundary layer and endplate surface topography are associated with brittleness of human whole vertebral bodies

Stress magnitude and variability as estimated from large scale finite element (FE) analyses have been associated with compressive strength of human vertebral cancellous cores but these relationships have not been explored for whole vertebral bodies. In this study, the objectives were to investigate the relationship of FE-calculated stress distribution parameters with experimentally determined strength, stiffness, and displacement based ductility measures in human whole vertebral bodies, investigate the effect of endplate loading conditions on vertebral stiffness, strength, and ductility and test the hypothesis that endplate topography affects vertebral ductility and stress distributions. Eighteen vertebral bodies (T6-L3 levels; 4 female and 5 male cadavers, aged 40-98 years) were scanned using a flat-panel CT system and followed with axial compression testing with Woods metal as filler material to maintain flat boundaries between load plates and specimens. FE models were constructed using reconstructed CT images and filler material was added digitally. Two different FE models with different filler material modulus simulating Woods metal and intervertebral disc (W-layer and D-layer models) were used. Element material modulus to cancellous bone was based on image gray value. Average, standard deviation, and coefficient of variation of von Mises stress in vertebral bone for W-layer and D-layer models and also the ratios of FE parameters from the two models (W/D) were calculated. Inferior and superior endplate surface topographical distribution parameters were calculated. Experimental stiffness, maximum load and work to fracture had the highest correlation with FE-calculated stiffness while experimental ductility measures had highest correlations with FE-calculated average von Mises stress and W-layer to D-layer stiffness ratio. Endplate topography of the vertebra was also associated with its structural ductility and the distribution parameter that best explained this association was kurtosis of inferior endplate topography. Our results indicate that endplate topography variations may provide insight into the mechanisms responsible for vertebral fractures.

Human cancellous bone from T12–L1 vertebrae has unique microstructural and trabecular shear stress properties

Increase of trabecular stress variability with loss of bone mass has been implicated as a mechanism for increased cancellous bone fragility with age and disease. In the current study, a previous observation that trabecular shear stress estimates vary along the human spine such that the cancellous tissue from the thoracic 12 (T12)-lumbar 1 (L1) junction experiences the highest trabecular stresses for a given load was tested as a formal hypothesis using multiple human spines. Thoracic 4, T5, T7, T9, T10, T12, L1, L2, L4 and L5 vertebrae from 10 human cadaver spines were examined. One specimen in the central anterior region was cored in the supero-inferior (SI) direction and another in the postero-lateral region was cored in the transverse (TR) direction from each vertebra. Micro-CT-based large-scale finite element models were constructed for each specimen and compression in the long axis of the cylindrical specimens was simulated. Cancellous bone modulus and the mean, the standard deviation, variability and amplification of trabecular von Mises stresses were computed. Bone volume fraction, trabecular number, trabecular thickness, trabecular separation, connectivity density and degree of anisotropy were calculated using 3D stereology. The results were analyzed using a mixed model in which spine level was modeled using a quadratic polynomial. The maximum of trabecular shear stress amplification and minimum of bone volume fraction were found in the cancellous tissue from the T12-L1 location when results from the samples of the same vertebra were averaged. When groups were separated, microstructure and trabecular stresses varied with spine level, extrema being at the T12-L1 levels, for the TR specimens only. SI/TR ratio of measured parameters also had quadratic relationships with spine level, the extrema being located at T12-L1 levels for most parameters. For microstructural parameters, these ratios approached to a value of one at the T12-L1 level, suggesting that T12-L1 vertebrae have more uniform cancellous tissue properties than other levels. The mean intercept length in the secondary principal direction of trabecular orientation could account for the variation of all mechanical parameters with spine level. Our results support that cancellous tissue from T12-L1 levels is unique and may explain, in part, the higher incidence of vertebral fractures at these levels.

Inhibition of prostate cancer-meditated osteoblastic bone lesions by the low-calcemic analog 1α-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D3

Prostate cancer metastasizes almost exclusively into the bone whereby it induces primarily an osteoblastic response. Non-calcemic vitamin D analogs have been shown to inhibit proliferation of prostate cancer cells in culture and inhibit their growth as subcutaneous xenografts in mice. However, their effect on prostate cancer cell growth in the bone has not been examined. In the present study, we inoculated the osteoblastic prostate cancer cell line MDA-PCa 2b into the bone of male SCID mice and examined the effect of the low-calcemic hybrid analog 1alpha-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D(3) (JK-1626-2) on their ability to induce bone lesions. We found that 7 weeks after inoculation of MDA-PCa 2b cells, 90% of the mice in the vehicle-treated group had significant bone lesions that were detectable by micro-computed tomography and characterized by thickening of the cortical bone and ossification of the epiphysis. Only 30% of the mice in the analog-treated group (daily injections of 4microg/kg, 5 days/week for up to 7 weeks) had detectable bone lesions. Histological examination of the decalcified tumor-bearing bones has shown that tumor cells completely replaced the bone marrow in the diaphysis, and destroyed the trabecular bone in the metaphysis in 90% of the vehicle-treated mice. In contrast, the metaphysis of 60% of analog-treated mice appeared normal, although tumor cells were still found in the diaphysis of 70% of the bones in the analog-treated group. There was no evidence of hypercalcemia in any of the analog-treated mice. In a co-culture, MDA-PCa 2b cells induced a profound mitogenic response in osteoblasts followed by enhanced differentiation. However, in the presence of the analog the mitogenic response of the osteoblasts to the malignant cells was significantly attenuated. These experiments led to the hypothesis that, in vivo, JK-1626-2 prevented the metastatic bone lesions by inhibiting the mitogenic response of osteoblasts to growth factors produced by MDA-PCa 2b cells.

Quantification of Mineralized Bone Response to Prostate Cancer by Noninvasive In Vivo μCT and Non-Destructive Ex Vivo μCT and DXA in a Mouse Model

Background To compare nondestructive in vivo and ex vivo micro-computed tomography (μCT) and ex vivo dual-energy-X-ray-absorptiometry (DXA) in characterizing mineralized cortical and trabecular bone response to prostate cancer involving the skeleton in a mouse model. Methodology/Principal Findings In vivo μCT was performed before and 10 weeks after implantation of human prostate cancer cells (MDA-PCa-2b) or vehicle into SCID mouse femora. After resection, femora were imaged by nondestructive ex vivo specimen μCT at three voxel sizes (31 µ, 16 µ, 8 µ) and DXA, and then sectioned for histomorphometric analysis of mineralized bone. Bone mineral density (BMD), trabecular parameters (number, TbN; separation, TbSp; thickness, TbTh) and mineralized bone volume/total bone volume (BV/TV) were compared and correlated among imaging methods and histomorphometry. Statistical tests were considered significant if P<0.05. Ten weeks post inoculation, diaphyseal BMD increased in the femur with tumor compared to the opposite femur by all modalities (p<0.005, n = 11). Diaphyseal BMD by in vivo μCT correlated with ex vivo 31 and 16 µm μCT and histomorphometry BV/TV (r = 0.91–0.94, P<0.001, n = 11). DXA BMD correlated less with bone histomorphometry (r = 0.73, P<0.001, n = 11) and DXA did not distinguish trabeculae from cortex. By in vivo and ex vivo μCT, trabecular BMD decreased (P<0.05, n = 11) as opposed to the cortex. Unlike BMD, trabecular morphologic parameters were threshold-dependent and when using “fixed-optimal-thresholds,” all except TbTh demonstrated trabecular loss with tumor and correlated with histomorphometry (r = 0.73–0.90, P<0.05, n = 11). Conclusions/Significance Prostate cancer involving the skeleton can elicit a host bone response that differentially affects the cortex compared to trabeculae and that can be quantified noninvasively in vivo and nondestructively ex vivo.

Contributions of Severe Burn and Disuse to Bone Structure and Strength in Rats

Burn and disuse results in metabolic and bone changes associated with substantial and sustained bone loss. Such loss can lead to an increased fracture incidence and osteopenia. We studied the independent effects of burn and disuse on bone morphology, composition and strength, and microstructure of the bone alterations 14days after injury. Sprague-Dawley rats were randomized into four groups: Sham/Ambulatory (SA), Burn/Ambulatory (BA), Sham/Hindlimb Unloaded (SH) and Burn/Hindlimb Unloaded (BH). Burn groups received a 40% total body surface area full-thickness scald burn. Disuse by hindlimb unloading was initiated immediately following injury. Bone turnover was determined in plasma and urine. Femur biomechanical parameters were measured by three-point bending tests and bone microarchitecture was determined by micro-computed tomography (uCT). On day 14, a significant reduction in body mass was observed as a result of burn, disuse and a combination of both. In terms of bone health, disuse alone and in combination affected femur weight, length and bone mineral content. Bending failure energy, an index of femur strength, was significantly reduced in all groups and maximum bending stress was lower when burn and disuse were combined. Osteocalcin was reduced in BA compared to the other groups, indicating influence of burn. The reductions observed in femur weight, BMC, biomechanical parameters and indices of bone formation are primarily responses to the combination of burn and disuse. These results offer insight into bone degradation following severe injury and disuse.

Engineered cartilage heals skull defects

INTRODUCTION The purposes of this study were to differentiate embryonic limb bud cells into cartilage, characterize the nodules produced, and determine their ability to heal a mouse skull defect. METHODS Aggregated mouse limb bud cells (E12-E12.5), cultured in a bioreactor for 3 weeks, were analyzed by histology or implanted in 6 skull defects. Six controls had no implants. The mice were scanned with microcomputed tomography weekly. At 2 and 4 weeks, a mouse from each group was killed, and the defect region was prepared for histology. RESULTS Chondrocytes in nodules were mainly hypertrophic. About 90% of the nodules mineralized. BrdU staining showed dividing cells in the perichondrium. Microcomputed tomography scans showed increasing minerals in implanted nodules that completely filled the defect by 6 weeks; defects in the control mice were not healed by then. At 2 and 4 weeks, the control skull sections showed only a thin bony layer over the defect. At 2 weeks, bone and cartilage filled the defects with implants, and the implants were well integrated with the adjacent cortical bone. At 4 weeks, the implant had turned almost entirely into bone. CONCLUSIONS Cartilage differentiated in the bioreactor and facilitated healing when implanted into a defect. Engineering cartilage to replace bone is an alternative to current methods of bone grafting.

Micro-CT-Based Large Scale Linear Finite Element Models Predict the Strength of Human Thoracic and Lumbar Vertebral Bodies

Vertebral fractures are among the most common and debilitating fractures. Structural organization of cancellous and cortical bone in a vertebra and their local properties are important factors that determine the strength of a vertebra. Linear finite element models utilizing Quantitative Computed Tomography (QCT) images have proven useful for predicting vertebral strength and are potentially useful in predicting risk of fracture in a clinical setting [1]. However, the amount of architectural detail in these models is not sufficient for studying trabecular stress and strains, and their relationship with the microscopic structure, which is important for understanding the mechanisms behind vertebral fragility.Copyright

SU‐FF‐I‐60: Image Quality Improvement Using a Custom Ventilator for Respiratory‐Gated Micro‐CT

Purpose: Acquiring Micro‐CT images of murine lungsin vivo requires respiratory gating to achieve optimal tissuecontrast at full inspiration. The use of a custom small‐animal ventilator and a specific acquisition technique has resulted in a dramatic improvement in image quality. Method and Materials: A commercially available small animal ventilator was customized for use with our commercially available micro‐CT scanner. The modified ventilator can hold the pressure in rodent lungs at a constant level (essentially perform a series of short breath‐holds at full inspiration), and can be programmed to trigger the scanner acquisition phase. The ventilator is set to cyclicly achieve a pre‐set pressure level of the anesthetic gas mixture (inspiration), trigger the x‐ray on phase of the scanner, acquire a single frame during the stable pressure phase, close the x‐ray shutter, and finally release the pressure of the anesthetic gas mixture (expiration) while the scanner rotates to the next view position, at which point the cycle repeats. Images can be reconstructed with either 90 or 45 micron isotropic voxels. Example images of several mice with lungtumors will be presented. Radiation dose is estimated to be about 0.26 Gy per scan session using this methodology. Results: This implementation has produced images with improved visibility of the tissue components, including airways and pulmonary blood vessels. The margins of individual lung lobes can be identified, which is very helpful when determining the location of a pathologic structure (lesion) for research purposes or subsequent necropsy. The improved image quality is also very helpful in the detection of small (0.3–0.5 mm diameter) lung lesions. Conclusion:Lungcancer research can benefit from extensive use of mouse models to investigate potential new treatment approaches. The use of improved non‐invasive imaging has positively impacted our lungcancer research program.