Nondestructive micro-computed tomography for biological imaging and quantification of scaffold-bone interaction in vivo

Scaffolds, also called bioscaffolds, are needed in all tissue engineering applications as carriers for cells and biochemical factors, as constructs providing appropriate mechanical conditions, or as a combination of the two. The aim of this paper is to present recent developments in micro-computed tomography (microCT) analyses of scaffolds. The focus will be on imaging and quantification aspects in bone research, and will deal with the assessment of scaffold architecture and how it interacts with bone tissue. We show that micro-architectural imaging is a nondestructive and noninvasive procedure that allows a precise three-dimensional (3D) measurement of scaffold architecture. Direct microCT-based image analysis allows to accurately quantify scaffold porosity, surface area, and 3D measures such as pore size, pore distribution, and strut thickness; furthermore, it allows for a precise measurement of bone growth into the scaffold and onto its surface. This methodology is useful for quality control of scaffold fabrication processes, to assess scaffold degradation kinetics, and to assess bone tissue response. Even more so, in combination with bioreactors or in vivo animal models, microCT allows to qualitatively and quantitatively assess the spatial and temporal mineralization of bone tissue formation in scaffolds; such longitudinal studies improve the assessment of bone response due to scaffold architecture. Computational models will be helpful in further analyses of these data in order to improve our understanding of mechanical and biochemical stimuli on bone formation, and are likely to provide valuable knowledge to optimize scaffold design.     

Impact of visceral adiposity measured by abdominal computed tomography on pulmonary function

Although an inverse relationship between abdominal adiposity and pulmonary function has been suggested, direct measurement of abdominal adipose tissue has rarely been attempted. Our object is to determine the impact of abdominal adiposity on pulmonary function by directly measuring abdominal adipose tissue with abdominal computed tomography (CT). In this cross-sectional study, we included never-smokers between the ages of 18 and 85 yr, who had undergone spirometry and abdominal adipose tissue analysis with CT scans during November 1, 2005 to October 31, 2009 as part of the comprehensive health examination. Among a total of 3,469 participants, 890 (25.7%) were male. The mean body mass index and waist circumference among males and females were 24.6 kg/m(2) and 87.8 cm and 23.0 kg/m(2) and 83.0 cm, respectively. Although total adipose tissue (TAT) of the abdomen in males (269.1 cm(2)) was similar to that in females (273.6 cm(2)), the ratio of visceral adipose tissue (VAT)/subcutaneous adipose tissue (SAT) was different; 0.99 in males and 0.50 in females. In males, TAT, SAT, and VAT were inversely associated with the absolute value of forced vital capacity (FVC), and TAT and VAT were inversely associated with forced expiratory volume in one second (FEV(1)). However, in females, TAT and VAT, but not SAT, were inversely associated with absolute FVC and FEV(1) values. In conclusion, the amount of abdominal adipose tissue directly measured using CT is inversely associated with lung function.     

Mapping the calcification of bovine pericardium in rat model by enhanced micro-computed tomography

The calcification initiation and progression of bioprosthetic heart valve were investigated in a rat model by enhanced micro-computed tomography, together with histologic study and scanning electron microscope analysis. The implantation data at early stage showed apparent dendritic patterns in the radiographic images for the glutaraldehyde-treated bovine pericardium and this dendritic pattern was verified to be associated with the vessel distribution in the tissue. Histologic study and scanning electron microscope analysis both indicated that the calcium deposits in the pericardium vessels regions were more grievous than those scattered in the collagen fibers in the first two weeks after implantation. Subsequently, calcification spreaded and the entire sample was severely calcified in 60 days.     

In vivo small animal imaging: current status and future prospects

The use of small animal models in basic and preclinical sciences constitutes an integral part of testing new pharmaceutical agents prior to commercial translation to clinical practice. Whole-body small animal imaging is a particularly elegant and cost-effective experimental platform for the timely validation and commercialization of novel agents from the bench to the bedside. Biomedical imaging is now listed along with genomics, proteomics, and metabolomics as an integral part of biological and medical sciences. Miniaturized versions of clinical diagnostic modalities, including but not limited to microcomputed tomography, micromagnetic resonance tomography, microsingle-photon-emission tomography, micropositron-emission tomography, optical imaging, digital angiography, and ultrasound, have all greatly improved our investigative abilities to longitudinally study various experimental models of human disease in mice and rodents. After an exhaustive literature search, the authors present a concise and critical review of in vivo small animal imaging, focusing on currently available modalities as well as emerging imaging technologies on one side and molecularly targeted contrast agents on the other. Aforementioned scientific topics are analyzed in the context of cancer angiogenesis and innovative antiangiogenic strategies under-the-way to the clinic. Proposed hybrid approaches for diagnosis and targeted site-specific therapy are highlighted to offer an intriguing glimpse of the future.     

In vivo quantification of cell-polymer interactions

An in vivo rat model was developed to determine cell-polymer interactions under physiological conditions. Microporous tubular grafts, made of polytetrafluoroethylene, a polyetherurethane, a polyesterurethane and also a modified polyetherurethane were implanted intraperitoneally in rats. The grafts were filled with cultured rat smooth muscle cells prior to implantation. At t = 0, 2 and 48 h, the grafts were evaluated macroscopically and also prepared for light microscopy and for cell count of their contents. At t = 0 no cellular attachment was observed on the lumenal side of the capsules. At t = 2 h a monolayer of smooth muscle cells could be observed on all materials except PTFE, on which only small patches of cells were observed. At t = 48 h a multilayer of cells was seen on all materials except PTFE. Cell counts at 48 h demonstrated no multiplication in the PTFE graft but a 1.4, 2.3 and 2.0-fold multiplication in the polyetherurethane, polyesterurethane and the modified polyurethane grafts respectively. These in vivo results show a clear linear relationship with our in vitro results in which it has been proved that cell spreading increased with increasing substratum surface free energy. This rat model allows the study of cell-polymer interactions in vivo, in a standardized way, under controlled physiological conditions.     

Radiation effects on bone architecture in mice and rats resulting from in vivo micro-computed tomography scanning

Recently established techniques for performing in vivo micro-computed tomography (micro-CT) provide the capability of monitoring bone changes in a living animal at various points in time. However, radiation exposure from repeated micro-CT scans may have an effect on skeletal growth in normal or disease-model animals. The purpose of this study is to test a high resolution ( approximately 10mum) in vivo micro-CT protocol on mice and rats used for bone research to understand the impact of micro-CT radiation exposure on bone architecture. Ovariectomy (OVX) or sham-OVX surgery was performed on groups (n=6-8/group) of 12-week-old C3H/HeJ, C57BL/6J, and BALB/cByJ mice, and one strain of rat (Wistar, retired breeders). The right proximal tibiae were scanned at weekly intervals while the contralateral left limbs were not scanned until the endpoint of the protocol. Trabecular and cortical bone morphology was compared between radiated and non-radiated limbs at the endpoint to quantify the radiation effect. No effects of radiation were observed in OVX or sham rats. Lower trabecular bone volume was observed in the radiated limbs (-8 to -20% relative to non-radiated limb) of all mice groups except sham BALB/cByJ mice and normal control C57BL/6J mice, however, the observed effects were much less than the observed effects of ovariectomy ( approximately 40-50% total bone volume reduction, depending on mouse strain), and no interactions between radiation and OVX treatment were observed (p>0.2). Using an internal non-radiated control within each animal is a potential method to elucidate the effect of radiation exposure for any in vivo protocol. Thus, although in vivo micro-CT is a valuable tool for bone-related research, the impact of radiation in skeletally immature mice should be considered, particularly for strains with low bone volume at the measured site.     

Improved quantification of small objects in near-infrared diffuse optical tomography

Diffuse optical tomography allows quantification of hemoglobin, oxygen saturation, and water in tissue, and the fidelity in this quantification is dependent on the accuracy of optical properties determined during image reconstruction. In this study, a three-step algorithm is proposed and validated that uses the standard Newton minimization with Levenberg-Marquardt regularization as the first step. The second step is a modification to the existing algorithm using a two-parameter regularization to allow lower damping in a region of interest as compared to background. This second stage allows the recovery of the actual size of an inclusion. A region-based reconstruction is the final third step, which uses the estimated size and position information from step 2 to yield quantitatively accurate average values for the optical parameters. The algorithm is tested on simulated and experimental data and is found to be insensitive to object contrast and position. The percentage error between the true and the average recovered value for the absorption coefficient in test images is reduced from 47 to 27% for a 10-mm inclusion, from 38 to 13% for a 15-mm anomaly, and from 28 to 5.5% for a 20-mm heterogeneity. Simulated data with absorbing and scattering heterogeneities of 15 mm diam located in different positions show recovery with less than 15% error in absorption and 6% error in reduced scattering coefficients. The algorithm is successfully applied to clinical data from a subject with a breast abnormality to yield quantitatively increased absorption coefficients, which enhances the contrast to 3.8 compared to 1.23 previously.     

Developing a small animal model of extracorporeal circulation

To identify nonthrombogenic devices to be used in extracorporeal circulation (ECC), an efficient, small animal model is required. Initially, a venovenous (VV) model in rabbits was designed for this purpose and was a good representation of ECC. Technical difficulties in the VV model led to the development of a more simplistic arteriovenous (AV) model. Anesthetized, tracheotomized, 3-kg rabbits were used for both models. Circuits were constructed of PVC tubing. The VV model used 8-Fr umbilical artery catheters for both drainage and reinfusion, and the AV model used a 14-GA angiocatheter for carotid artery access and a 10-Fr thoracic catheter for venous access. The AV model included a chamber to mimic oxygenator or filter modeling. Hourly measurements included blood gases, platelet counts, and fibrinogen levels for the 4-hour studies. The VV ECC groups demonstrated platelet consumption like that seen in the clinical arena. The AV model demonstrated the same with or without additional surface area within the chamber. The AV model was deemed to be superior due to its simplicity, ability for filter modeling, and decrease in intensive monitoring. However, both models are excellent designs for nonthrombogenic surface testing.     

Role of oxidative stress in animal model of visceral pain

Reactive oxygen species play an important role both in physiological and pathophysiological reactions. The aim of this study was to determine the role of free radicals and antioxidants in the development of visceral pain. Visceral pain was produced by colorectal distension (CRD) in adult rats. CRD was caused by insertion of a lubricated latex balloon into the descending colon and rectum followed by inflation to 80 mm Hg for 10 min. During CRD, visceral pain was rated on 0–3.5 point scale. Oxidative stress was determined indirectly by measurement of free radical scavenging enzymes (glutathione peroxidase (GPx) and superoxide dismutase (SOD)) in the blood, liver and brain. Following CRD we observed (1) all rats expressed signs of visceral pain (overall rating was 1.83), (2) SOD and GPx levels were increased in the liver and blood, and decreased in the brain samples and (3) administration of the antioxidant Trolox, a water-soluble derivate of vitamin E, prior to CRD, prevented SOD and GPx changes in the liver, blood and brain, but did not affect pain scores. It was concluded, that CRD as a model of visceral pain, increases oxidative stress in animals, which could be prevented by prior administration of antioxidants; however, antioxidants did not attenuate signs of visceral pain caused by CRD.     

Synchrotron-based intravenous cerebral angiography in a small animal model

K-edge digital subtraction angiography (KEDSA), a recently developed synchrotron-based technique, utilizes monochromatic radiation and allows acquisition of high-quality angiography images after intravenous administration of contrast agent. We tested KEDSA for its suitability for intravenous cerebral angiography in an animal model. Adult male New Zealand rabbits were subjected to either angiography with conventional x-ray equipment or synchrotron-based intravenous KEDSA, using an iodine-based contrast agent. Angiography with conventional x-ray equipment after intra-arterial administration of contrast agent demonstrated the major intracranial vessels but no smaller branches. KEDSA was able to visualize the major intracranial vessels as well as smaller branches in both radiography mode (planar images) and tomography mode. Visualization was achieved with as little as 0.5 ml kg-1 of iodinated contrast material. We were able to obtain excellent visualization of the cerebral vasculature in an animal model using intravenous injection of contrast material, using synchrotron-based KEDSA.     

Contrast-enhanced micro-computed tomography of compartment and time-dependent changes in femoral cartilage and subchondral plate in a murine model of osteoarthritis

A lack of understanding of the mechanisms underlying osteoarthritis (OA) progression limits the development of effective long-term treatments. Quantitatively tracking spatiotemporal patterns of cartilage and bone degeneration is critical for assessment of more appropriately targeted OA therapies. In this study, we use contrast-enhanced micro-computed tomography (μCT) to establish a timeline of subchondral plate (SCP) and cartilage changes in the murine femur after destabilization of the medial meniscus (DMM). We performed DMM or sham surgery in 10–12-week-old male C57Bl/6J mice. Femora were imaged using μCT after 0, 2, 4, or 8 weeks. Cartilage-optimized scans were performed after immersion in contrast agent CA4+. Bone mineral density distribution (BMDD), cartilage attenuation, SCP, and cartilage thickness and volume were measured, including lateral and medial femoral condyle and patellar groove compartments. As early as 2 weeks post-DMM, cartilage thickness significantly increased and cartilage attenuation, SCP volume, and BMDD mean significantly decreased. Trends in cartilage and SCP metrics within each joint compartment reflected those seen in global measurements, and both BMDD and SCP thickness were consistently greater in the lateral and medial condyles than the patellar groove. Sham surgery also resulted in significant changes to SCP and cartilage metrics, highlighting a potential limitation of using surgical models to study tissue morphology or composition changes during OA progression. Contrast-enhanced μCT analysis is an effective tool to monitor changes in morphology and composition of cartilage, and when combined with bone-optimized μCT, can be used to assess the progression of degenerative changes after joint injury.     

Optimizing in vivo small animal Cerenkov luminescence imaging

In vivo Cerenkov luminescence imaging is a rapidly growing molecular imaging research field based on the detection of Cerenkov radiation induced by beta particles when traveling though biological tissues. We investigated theoretically the possibility of enhancing the number of the detected Cerenkov photons in the near infrared (NIR) region of the spectrum. The analysis is based on applying a photon propagation diffusion model to Cerenkov photons in the tissue. Results show that despite the smaller number of Cerenkov photons in the NIR region, the fraction exiting the tissues is greater than in the visible range, and thus, a charge-coupled device detector optimized for the NIR range will allow to obtain a higher signal. The comparison was performed considering Cerenkov point sources located at different depths inside the animal. We concluded that the improvement can be up to 35% and is more significant when the Cerenkov source to be imaged is located deeper inside the animal.     

In vivo effects of apoptosis in asthma examined by a murine model

BACKGROUND: One of the characteristic features of bronchial asthma is the accumulation of various inflammatory cells, predominantly eosinophils, at the subepithelial region beneath the basement membrane of the airway. Apoptosis is a form of physiological cell death, through which the cellular contents including biologically active substances are kept in the cell membrane and are removed without their harmful effects. So, attempts were made to clarify whether the induction of apoptosis is beneficial in asthma by using a murine model with ovalbumin (OA) as responsible allergen. METHODS: A/J mice, which are genetically predisposed to be hyperresponsive to acetylcholine, were immunized with OA and alum, accompanied by OA inhalation for 2 weeks, during which some of the mice were also treated with either anti-Fas monoclonal antibody or sham control hamster IgG intranasally. Airway responsiveness to acetylcholine was then analyzed by measuring airway resistance with a body plethysmograph box. Apoptosis was assessed by propidium iodide and TUNEL staining. RESULTS: Inhalation of OA increased both airway responsiveness to acetylcholine and the number of cells, mostly eosinophils, infiltrated into the airway. Administration of anti-Fas antibody induced apoptosis in the infiltrated eosinophils and abolished augmentation of airway hyperresponsiveness caused by OA inhalation. CONCLUSION: Induction of apoptosis in proinflammatory cells including eosinophils at the airway may have a beneficial effect on suppressing airway hyperresponsiveness.     

In vivo activation of complement by IgA in a rat model.

In this study we investigated the capacity of rat IgA to activate complement (C) in vivo in a rat model. Rat monomeric (m-), dimeric (d-) and polymeric (p-) IgA MoAbs were injected intravenously and assessed for deposition of C3 and C4 on IgA. By ELISA it was shown that both d- and p-IgA bound C3 whereas no binding of C3 by m-IgA was observed. Polymeric IgA was more efficient in binding of C3 as compared with d-IgA. However, in haemolytic assays no consistent decrease of plasma complement levels was observed except for dimeric IgA which induced a marginal consumption of AP50. When rats were pre-treated with cobra venom factor (CVF) to deplete C3, no C3 deposition was found on m-, d- or p-IgA. Neither m- nor d- or p-IgA was able to bind C4 in vivo. In agreement with the results described above, large sized polymeric IgA was shown to be taken up by Kupffer cells (KC) together with C3. No C3 was detected when rats were depleted of C using CVF. Taken together, the experimental data suggest that d- and p-IgA are able to activate C via the alternative pathway in vivo.     

Association between type 2 diabetes genetic susceptibility loci and visceral and subcutaneous fat area as determined by computed tomography

Visceral fat accumulation has an important role in the development of several metabolic disorders, such as type 2 diabetes, dyslipidemia and hypertension. New genetic loci that contribute to the development of type 2 diabetes have been identified by genome-wide association studies. To examine the association of type 2 diabetes susceptibility loci and visceral fat accumulation, we genotyped 1279 Japanese subjects (556 men and 723 women), who underwent computed tomography for measurements of visceral fat area (VFA) and subcutaneous fat area (SFA) for the following single-nucleotide polymorphisms (SNPs): NOTCH2 rs10923931, THADA rs7578597, PPARG rs1801282, ADAMTS9 rs4607103, IGF2BP2 rs1470579, VEGFA rs9472138, JAZF1 rs864745, CDKN2A/CDKN2B rs564398 and rs10811661, HHEX rs1111875 and rs5015480, TCF7L2 rs7901695, KCNQ1 rs2237892, KCNJ11 rs5215 and rs5219, EXT2 rs1113132, rs11037909, and rs3740878, MTNR1B rs10830963, DCD rs1153188, TSPAN8/LGR5 rs7961581, and FTO rs8050136 and rs9939609. None of the above SNPs were significantly associated with VFA. The FTO rs8050136 and rs9939609 risk alleles exhibited significant associations with body mass index (BMI; P=0.00088 and P=0.0010, respectively) and SFA (P=0.00013 and P=0.00017, respectively). No other SNPs were significantly associated with BMI or SFA. Our results suggest that two SNPs in the FTO gene are associated with subcutaneous fat accumulation. The contributions of other SNPs are inconclusive because of a limitation of the sample power.     

Hyperspectral and multispectral bioluminescence optical tomography for small animal imaging

For bioluminescence imaging studies in small animals, it is important to be able to accurately localize the three-dimensional (3D) distribution of the underlying bioluminescent source. The spectrum of light produced by the source that escapes the subject varies with the depth of the emission source because of the wavelength-dependence of the optical properties of tissue. Consequently, multispectral or hyperspectral data acquisition should help in the 3D localization of deep sources. In this paper, we describe a framework for fully 3D bioluminescence tomographic image acquisition and reconstruction that exploits spectral information. We describe regularized tomographic reconstruction techniques that use semi-infinite slab or FEM-based diffusion approximations of photon transport through turbid media. Singular value decomposition analysis was used for data dimensionality reduction and to illustrate the advantage of using hyperspectral rather than achromatic data. Simulation studies in an atlas-mouse geometry indicated that sub-millimeter resolution may be attainable given accurate knowledge of the optical properties of the animal. A fixed arrangement of mirrors and a single CCD camera were used for simultaneous acquisition of multispectral imaging data over most of the surface of the animal. Phantom studies conducted using this system demonstrated our ability to accurately localize deep point-like sources and show that a resolution of 1.5 to 2.2 mm for depths up to 6 mm can be achieved. We also include an in vivo study of a mouse with a brain tumour expressing firefly luciferase. Co-registration of the reconstructed 3D bioluminescent image with magnetic resonance images indicated good anatomical localization of the tumour.     

Bioluminescent and micro-computed tomography imaging of bone repair induced by fibrin-binding growth factors

In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (μCT) to evaluate bone regeneration and high-resolution μCT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3 weeks after implantation. Results from μCT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs.     

In vivo quantification of lead in bone with a portable x-ray fluorescence system--methodology and feasibility

This study was conducted to investigate the methodology and feasibility of developing a portable x-ray fluorescence (XRF) technology to quantify lead (Pb) in bone in vivo. A portable XRF device was set up and optimal settings of voltage, current, and filter combination for bone lead quantification were selected to achieve the lowest detection limit. The minimum radiation dose delivered to the subject was calculated by Monte Carlo simulations. An ultrasound device was used to measure soft tissue thickness to account for signal attenuation, and an alternative method to obtain soft tissue thickness from the XRF spectrum was developed and shown to be equivalent to the ultrasound measurements (intraclass correlation coefficient, ICC = 0.82). We tested the correlation of in vivo bone lead concentrations between the standard KXRF technology and the portable XRF technology. There was a significant correlation between the bone lead concentrations obtained from the standard KXRF technology and those obtained from the portable XRF technology (ICC = 0.65). The detection limit for the portable XRF device was about 8.4 ppm with 2 mm soft tissue thickness. The entrance skin dose delivered to the human subject was about 13 mSv and the total body effective dose was about 1.5 μSv and should pose minimal radiation risk. In conclusion, portable XRF technology can be used for in vivo bone lead measurement with sensitivity comparable to the KXRF technology and good correlation with KXRF measurements.