Quantitative measures of bone shape, cartilage morphometry and joint alignment are associated with disease in an ACLT and MMx rat model of osteoarthritis.

Multi-scale, subject-specific quantitative methods to characterize and monitor osteoarthritis in animal models and therapeutic treatments could help reveal causal relationships in disease development and distinguish treatment strategies. In this work, we demonstrate a reproducible and sensitive quantitative image analysis to characterize bone, cartilage and joint measures describing a rat model of post-traumatic osteoarthritis. Eleven 3-month-old male Wistar rats underwent medial anterior cruciate ligament (ACL) transection and medial meniscectomy on the right knee to destabilise the right tibiofemoral joint. They were sacrificed 6 weeks post-surgery and a silicon-based micro-bead contrast agent was injected in the joint space, before scanning with micro-computed tomography (microCT). Subsequently, 3D quantitative morphometric analysis (QMA), previously developed for rabbit joints, was performed. This included cartilage, subchondral cortical and epiphyseal bone measures, as well as novel tibiofemoral joint metrics. Semi-quantitative evaluation was performed on matching two-dimensional (2D) histology and microCT images. Reproducibility of the QMA was tested on eleven age-matched additional joints. The results indicate the QMA method is accurate and reproducible and that microCT-derived cartilage measurements are valid for the analysis of rat joints. The pathologic changes caused by transection of the ACL and medial meniscectomy were reflected in measurements of bone shape, cartilage morphology, and joint alignment. Furthermore, we were able to identify model-specific predictive parameters based on morphometric parameters measured with the QMA.

Implementation and application of a Monte Carlo model for an in vivo micro computed tomography system.

Micro computed tomography (µCT) scanners are used to create high-resolution images and to quantify properties of the scanned objects. While modern µCT scanners benefit from the cone beam geometry, they are compromised by scatter radiation. This work aims to develop a Monte Carlo (MC) model of a µCT scanner in order to characterize the scatter radiation in the detector plane. The EGS++ framework with the MC code EGSnrc was used to simulate the particle transport through the main components of the XtremeCT (SCANCO Medical AG, Switzerland). The developed MC model was based on specific information of the manufacturer and was validated against measurements. The primary and the scatter radiation were analyzed and by implementing a dedicated tracing method, the scatter radiation was subdivided into different scatter components. The comparisons of measured and simulated transmission values for different absorber and filter combinations result in a mean difference of 0.2% ±â€¯1.4%, with a maximal local difference of 3.4%. The reconstructed image of the phantom based on measurements agrees well with the image reconstructed using the MC model. The local contribution of scattered radiation is up to 10% of the total radiation in the detector plane and most of the scattered particles result from interactions in the scanned object. The MC simulations show that scatter radiation contains information about the structure of the object. In conclusion, a MC model for a µCT scanner was successfully validated and applied to analyze the characteristics of the scatter radiation for a µCT scanner.

Three-Dimensional Quantitative Morphometric Analysis (QMA) for In Situ Joint and Tissue Assessment of Osteoarthritis in a Preclinical Rabbit Disease Model.

This work utilises advances in multi-tissue imaging, and incorporates new metrics which define in situ joint changes and individual tissue changes in osteoarthritis (OA). The aims are to (1) demonstrate a protocol for processing intact animal joints for microCT to visualise relevant joint, bone and cartilage structures for understanding OA in a preclinical rabbit model, and (2) introduce a comprehensive three-dimensional (3D) quantitative morphometric analysis (QMA), including an assessment of reproducibility. Sixteen rabbit joints with and without transection of the anterior cruciate ligament were scanned with microCT and contrast agents, and processed for histology. Semi-quantitative evaluation was performed on matching two-dimensional (2D) histology and microCT images. Subsequently, 3D QMA was performed; including measures of cartilage, subchondral cortical and epiphyseal bone, and novel tibio-femoral joint metrics. Reproducibility of the QMA was tested on seven additional joints. A significant correlation was observed in cartilage thickness from matching histology-microCT pairs. The lateral compartment of operated joints had larger joint space width, thicker femoral cartilage and reduced bone volume, while osteophytes could be detected quantitatively. Measures between the in situ tibia and femur indicated an altered loading scenario. High measurement reproducibility was observed for all new parameters; with ICC ranging from 0.754 to 0.998. In conclusion, this study provides a novel 3D QMA to quantify macro and micro tissue measures in the joint of a rabbit OA model. New metrics were established consisting of: an angle to quantitatively measure osteophytes (σ), an angle to indicate erosion between the lateral and medial femoral condyles (ρ), a vector defining altered angulation (λ, α, ß, γ) and a twist angle (τ) measuring instability and tissue degeneration between the femur and tibia, a length measure of joint space width (JSW), and a slope and intercept (m, Χ) of joint contact to demonstrate altered loading with disease progression, as well as traditional bone and cartilage and histo-morphometry measures. We demonstrate correlation of microCT and histology, sensitive discrimination of OA change and robust reproducibility.

Quantification of lower leg arterial calcifications by high-resolution peripheral quantitative computed tomography.

Vascular calcifications and bone health seem to be etiologically linked via common risk factors such as aging and subclinical chronic inflammation. Epidemiologic studies have shown significant associations between low bone mineral density (BMD), fragility fractures and calcifications of the coronary arteries and the abdominal aorta. In the last decade, high-resolution peripheral quantitative computed tomography (HR-pQCT) has emerged as in-vivo research tool for the assessment of peripheral bone geometry, density, and microarchitecture. Although vascular calcifications are frequently observed as incidental findings in HR-pQCT scans, they have not yet been incorporated into quantitative HR-pQCT analyses. We developed a semi-automated algorithm to quantify lower leg arterial calcifications (LLACs), captured by HR-pQCT. The objective of our study was to determine validity and reliability of the LLAC measure. HR-pQCT scans were downscaled to a voxel size of 250µm. After subtraction of bone volumes from the scans, LLACs were detected and contoured by a semi-automated, dual-threshold seed-point segmentation. LLAC mass (in mg hydroxyapatite; HA) was calculated as the product of voxel-based calcification volume (mm(3)) and mean calcification density (mgHA/cm(3))/1000. To determine validity, we compared LLACs to coronary artery calcifications (CACs), as quantified by multi-detector computed tomography (MDCT) and Agatston scoring in forty-six patients on chronic hemodialysis. Moreover, we investigated associations of LLACs with age, time on dialysis, type-2 diabetes mellitus, history of stroke, and myocardial infarction. In a second step, we determined intra- and inter-reader reliability of the LLAC measure. In the validity study, LLACs were present (>0mgHA) in 76% of patients, 78% of patients had CACs (>0mgHA). Median LLAC was 6.65 (0.08-24.40)mgHA and median CAC as expressed by Agatston score was 266.3 (15.88-1877.28). We found a significant positive correlation between LLAC and CAC (rho=0.6; p<0.01). Dialysis patients with type-2 diabetes mellitus (DM; 35%) and history of stroke (13%) had higher median LLAC than patients without those conditions (DM 20.0 fold greater, p=0.006; Stroke 5.1 fold greater, p=0.047). LLAC was positively correlated with time on dialysis (rho=0.337, p=0.029), there was a trend towards a positive association of LLAC and age (rho=0.289, p=0.053). The reliability study yielded excellent intra- and inter-reader agreement of the LLAC measure (intra-reader ICC=0.999, 95% CI=0.998-1.000; inter-reader ICC=0.998, 95% CI=0.994-0.999). Our study indicates that the LLAC measure has good validity and excellent reliability. The use of HR-pQCT for the simultaneous evaluation of arterial calcifications, peripheral bone geometry, bone density, and bone microarchitecture should facilitate future research on osteo-vascular interactions and potential associations with cardiovascular events.

A correlative method for imaging identical regions of samples by micro-CT, light microscopy, and electron microscopy: imaging adipose tissue in a model system.

We present a method in which a precise region of interest within an intact organism is spatially mapped in three dimensions by non-invasive micro-computed X-ray tomography (micro-CT), then further evaluated by light microscopy (LM) and transmission electron microscopy (TEM). Tissues are prepared as if for TEM including osmium fixation, which imparts soft tissue contrast in the micro-CT due to its strong X-ray attenuation. This method may therefore be applied to embedded, archived TEM samples. Upon selection of a two-dimensional (2-D) projection from a region of interest (ROI) within the three-dimensional volume, the epoxy-embedded sample is oriented for microtomy so that the sectioning plane is aligned with the micro-CT projection. Registration is verified by overlaying LM images with 2-D micro-CT projections. Structures that are poorly resolved in the micro-CT may be evaluated at TEM resolution by observing the next serial ultrathin section, thereby accessing the same ROI by all three imaging techniques. We compare white adipose tissue within the forelimbs of mice harboring a lipid-altering mutation with their littermate controls. We demonstrate that individual osmium-stained lipid droplets as small as 15 µm and separated by as little as 35 µm may be discerned as separate entities in the micro-CT, validating this to be a high-resolution, non-destructive technique for evaluation of fat content.

Structural strain energy function applied to the ageing of the human aorta.

Stiffening of the aorta with progressing age leads to decrease of aortic compliance and thus to an increase of pulse pressure amplitude. Using a strain energy function (SEF) which takes into account the composition of the arterial wall, we have studied the evolution of key structural components of the human thoracic aorta using data obtained from the literature. The SEF takes into account the wavy nature of collagen, which upon gradual inflation of the blood vessel is assumed to straighten out and become engaged in bearing load. The engagement of the individual fibers is assumed to be distributed log-logistically. The use of a SEF enables the consideration of axial stretch (lambda(z)) and residual strain (opening angle) in the biomechanical analysis. Both lambda(z) and opening angle are known to change with age. Results obtained from applying the SEF to the measurements of aortic pressure-diameter curves indicate that the changes in aortic biomechanics with progressing age are not to be sought in the elastic constants of elastin and collagen or their volume fractions of the aortic wall but moreover in alterations of the collagen mesh arrangement and the waviness of the collagen fibers. In old subjects, the collagen fiber ensemble engages in load bearing much more abruptly than in young subjects. Reasons for this change in collagen fiber dynamics may include fiber waviness remodeling or cross-linkage by advanced glycation end-products (AGE). The abruptness of collagen fiber engagement is also the model parameter that is most responsible for the decreased compliance at progressed ages.

A constitutive formulation of arterial mechanics including vascular smooth muscle tone.

A pseudo-strain energy function (pseudo-SEF) describing the biomechanical properties of large conduit arteries under the influence of vascular smooth muscle (VSM) tone is proposed. In contrast to previous models that include the effects of smooth muscle contraction through generation of an active stress, in this study we consider the vascular muscle as a structural element whose contribution to load bearing is modulated by the contraction. This novel pseudo-SEF models not only arterial mechanics at maximal VSM contraction but also the myogenic contraction of the VSM in response to local increases in stretch. The proposed pseudo-SEF was verified with experimentally obtained pressure-radius curves and zero-stress state configurations from rat carotid arteries displaying distinct differences in VSM tone: arteries from normotensive rats displaying minimal VSM tone and arteries from hypertensive rats exhibiting significant VSM tone. The pressure-radius curves were measured in three different VSM states: fully relaxed, maximally contracted, and normal VSM tone. The model fitted the experimental data very well (r2 > 0.99) in both the normo- and hypertensive groups for all three states of VSM activation. The pseudo-SEF was used to illustrate the localized reduction of circumferential stress in the arterial wall due to normal VSM tone, suggesting that the proposed pseudo-SEF can be of general utility for describing stress distribution not only under passive VSM conditions, as most SEFs proposed so far, but also under physiological and pathological conditions with varying levels of VSM tone.

A strain energy function for arteries accounting for wall composition and structure.

Identification of a Strain Energy Function (SEF) is used when describing the complex mechanical properties of soft biological tissues such as the arterial wall. Classic SEFs, such as the one proposed by Chuong and Fung (J. Biomech. Eng. 105(3) (1983) 268), have been mostly phenomenological and neglect the particularities of the wall structure. A more structural model was proposed by Holzapfel et al. (J. Elasticity 61 (2000) 1-48.) when they included the characteristic angle at which the collagen fibers are helically wrapped, resulting in an excellent SEF for applications such as finite element modeling. We have expanded upon the idea of structural SEFs by including not only the wavy nature of the collagen but also the fraction of both elastin and collagen contained in the media, which can be determined by histology. The waviness of the collagen is assumed to be distributed log-logistically. In order to evaluate this novel SEF, we have used it to fit experimental data from inflation-extension tests performed on rat carotids. We have compared the results of the fit to the SEFs of Choung and Fung and Holzapfel et al. The novel SEF is found to behave similarly to that of Holzapfel et al., both succeed in describing the typical S-shaped pressure-radius curves with comparable quality of fit. The parameters of the novel SEF obtained from the fitting, bearing the physical meaning of the elastic modulus of collagen, the elastic modulus of elastin, the collagen waviness, and the collagen fiber angle, were compared to experimental data and discussed.

Effects of longitudinal stretch on VSM tone and distensibility of muscular conduit arteries.

With progressing age, large arteries diminish their longitudinal stretch, which in extreme cases results in tortuosity. Increased age is also associated with loss of vessel distensibility. We measured pressure-diameter curves from muscular porcine carotid arteries ex vivo at different longitudinal stretch ratios (lambda(z) = 1.4 and 1.8) and under different vascular smooth muscle (VSM) conditions (fully relaxed, normal VSM tone, and maximally contracted). Distensibility was found to be halved by decreasing longitudinal stretch from lambda(z) = 1.8 to 1.4 at physiological pressures. This counterintuitive observation is possible because highly nonlinear elastic modulus of the artery and anisotropic properties. Furthermore, a significantly larger basal VSM contraction was observed at lambda(z) = 1.8 than 1.4, although this was clearly not related to a myogenic response during inflation. This dependence of VSM tone to longitudinal stretch may have possible implications on the functional characteristics of the arterial wall.

Biomechanical adaptation of porcine carotid vascular smooth muscle to hypo and hypertension in vitro.

Previous research in arterial remodeling in response to changes in blood pressure seldom included both hyper- and hypotension. To compare the effects of low and high pressure on arterial remodeling and vascular smooth muscle tone and performance, we have utilized an in vitro model. Porcine carotid arteries were cultured for 3 days at 30 and 170mmHg and compared to controls cultured at 100mmHg for 1 and 3 days. On the first and last day of culture, pressure-diameter and pressure-wall thickness curves were measured under normal smooth muscle tone using a high-resolution ultrasonic device. Last-day experiments included measurements where vascular smooth muscle was contracted or totally relaxed. From the data wall cross-sectional area, Hudetz elastic modulus and a contraction index related to the diameter reduction under normal smooth muscle tone were calculated. We found that although wall cross-sectional area (indicating wall mass) did not change much, Hudetz elastic modulus was significantly reduced in the 3-day hypotension group. Inspection of the wall contraction index suggests that this is due to a reduction in the vascular smooth muscle tone. Further, the peak of contraction index was found to be shifted to higher pressures in the 3-day 170mmHg group. We conclude that vascular smooth muscle performance adapts to both hypo- and hypertension at short time scales and can alter the biomechanics of the vascular wall in vitro.