Recent Highlights in Materials Reliability Division

Members of the Biomaterials Metrology Project are working in conjunction with researchers at the University of Colorado and Children’s Hospital of Denver to characterize the development and pathophysiology of secondary pulmonary hypertension (PHT) in children. The high elevation of the Rocky Mountain region causes children born with heart defects to have a greater propensity for developing PHT, so it is an area of great concern for this region. Members of this collaborative team are addressing the role of fluid mechanics, biochemistry, pathology, histology, and the arterial wall mechanics during development and at maturation of the disease. It is our goal to develop new, less-invasive diagnostics that might hasten treatment and mitigate permanent damage. At the present time, diagnosis is sometimes delayed by two factors: a variety of conditions that can display similar clinical expression, and the reluctance of physicians to subject a child to catheterization unless it is clearly necessary.

The Pediatric Pulmonary Hypertension subproject is focusing on the dual goals of quantizing the mechanical properties and identifying the structural differences between normotensive and hypertensive pulmonary arteries (PAs). Our approach is to begin with a rat model to determine whether we can detect differences in the stress–strain and ultrasonic properties between conditions. We have chosen the Long–Evans breed of rats because they can be genetically modified so that the endothelin B receptor is disabled (knock-out or KO). The endothelin B receptor is responsible for activating the vasodilators prostacyclin and nitric oxide.

The test matrix includes testing controls (otherwise referred to as untreated wilds), monocrotaline-treated wilds, hypoxic wilds, hypoxic KOs, and perhaps monocrotaline-treated KOs. Monocrotaline is a synthetic chemical derived from a naturally occurring substance that produces an inflammatory response in the lungs that typically degenerates into PHT. The rats are injected with monocrotaline subcutaneously, and the disease is allowed to develop over four weeks before the arterial tissue is tested. To induce PHT with hypoxia, rats are held in a hypobaric chamber that simulates oxygen conditions at 5200 m above sea level for three weeks before testing. Specimens from the trunk, and the right and left main branches are tested. It is thought that these proximal arteries are likely to be used for clinical diagnosis as they are more readily imaged non-invasively.

A bubble-test configuration is employed to obtain stress–strain data (Figure 1). The specimen sits in a reservoir and is pressurized in 1.4 kPa (10 mm Hg) increments from the adventitial side with a physiological solution. Images of the bubble profile are collected at each pressure increment from six camera angles located 30° apart. These images are used to measure the changing length of the extruded bubble to calculate strain. The bubble is fitted as an ellipsoid to calculate the surface area to determine stress. The various camera angles allow us to account for anisotropic deformation. However, at this point, we are using the formulation for the stress at the apex of an ellipsoid of an isotropic material to approximate the stress. Models are nearing completion that will determine the elastic constants of the material from the experimental measurements based on established anisotropic constitutive relationships.

The results of the tests for each population are shown in Figures 2a–d. All values for strain were relative to the displacement measured at 1.4 kPa. Both the hypoxic wilds and the hypoxic knock-outs showed less strain at a given stress value than did the controls — the knock-outs more so than the wilds. It should also be pointed out that, in general, the hypoxic specimens were pressurized to equivalent pressures to the controls and monocrotaline treated specimens, and oftentimes 15–20 % higher. The thickness of the hypoxic specimens had a dramatic effect on the stress relationship. We found that the effect of the monocrotaline treatment on the wild population was unclear on the stress–strain relationship. We are in the process of identifying a reasonable mathematical model for the data so that statistical analyses may determine significance among the populations. Work has begun to correlate general trends in our pressure-displacement data to in vivo compliance data acquired by use of input impedance measurements in the catheterization laboratory.

Ultrasonic measurements have been performed and analyzed for three different rat populations (six controls, four wild hypoxics, and six KO hypoxics). We use a 50 MHz acoustic microscope in double-transmission mode with insonification in the radial direction of the PA wall. Thickness and speed of sound are determined by measuring changes in times-of-flight of ultrasonic signals through a reference path of a nutritive solution and a path substituted with the PA specimen. The slope of attenuation is determined by spectral analysis of these same ultrasonic signals. Current acoustic microscopy results are not yet fully understood. Models from the wild hypoxic rat exhibited an expected increase in speed of sound (up to 10 %) in the PA trunk compared with that of the controls, which is indicative of stiffening as a result of remodeling due to hypertension (see Figure 3a). However, the speed of sound in the KO hypoxic PAs does not appear to differ significantly from that of the controls. More marked increases (up to 100 %) were observed in slope of attenuation for the PA trunk for both hypoxic models as compared with controls (see Figure 3b). Smaller changes in the ultrasonic properties were observed for either the left or right PA branches. Continuing studies aim to investigate the effect of loading of tissue to mimic in vivo arterial pressures and to increase the population counts of the rat models to reduce the large standard deviations.

Figure 3: a) Comparison of the speed of sound and b) slope of attenuation in pulmonary branches for controls (n=6), hypoxic wilds (n=4), and hypoxic KO (n=6). Error bars represent standard deviations among the specimens.

The histology of the PAs is being evaluated to determine relationships between the structure and properties of the tissue, and to characterize remodeling of structure associated with increases in stiffness measured for patients with PHT. As shown in Figure 4, the extrapulmonary arteries have complex structures that provide the strength and elasticity required for the conduit function and environment.

Current observations indicate that the hypoxic knockouts have collagen deposition and vascularization in the adventitial layer adjacent to the medial layer. In Figure 4, for example, there is a gradient of apparent density through the adventitial layer, where control samples tend to have a structure more like that near the outside diameter of this example. Other measurements, such as elastic lamina thickness, sublayer thickness, and the size of the smooth muscle cells, are also being evaluated.

Figure 4: A cross-section of a pulmonary artery, taken just adjacent to the heart in a hypoxic knockout rat, showing the three major layers (endothelial, medial, and adventitial) characteristic of extrapulmonary arteries. The lumen (cavity in which the blood flows) is partially visible at the bottom edge of the figure. The endothelial layer, which is in contact with the blood, is a very thin layer adjacent to the lumen that ends at the first elastic lamella (black bands). The medial layer in this particular artery has about eight elastic lamella that separate it into about seven sublayers of smooth muscle. The adventitial layer is the third layer on the outside diameter of the artery. This layer is typically a fibrous low-density layer compared with the medial layer.

One of the advantages to conducting this work at NIST is our ability to apply different techniques to the same problem or issue, thereby gleaning a more complete understanding of the development of the pathophysiology of PHT and its effect on the pulmonary arterial mechanics. The elastic properties generated by the acoustic microscope provide the best opportunity for bridging the technical gap between clinical in vivo ultrasonic measurements and in vitro mechanical properties measured in the laboratory. The complementary work on the histology will help to corroborate the statistical significance in the mechanical properties observed among the populations. Moreover, the histological analysis can contribute to future treatments of PHT directed at the specific cell or biochemical expressions that result in reduced compliance of the arterial wall.

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