Background

Pulmonary Arterial Hypertension

Pulmonary hypertension (PAH) is a condition denoted by a mean pulmonary arterial pressure greater than 20 mmHg4. This increase in arterial pressure suggests that the heart must adapt to work under greater afterloads. As a result, the right ventricle (RV) of the heart undergoes structural and functional changes at different scales. The scale of interest sought to be observed via planar biaxial testing (to be described later) involves the loss of tissue contractility and changes in collagen fiber orientation. Currently, the National Institute of Health Registry on Primary Pulmonary Hypertension provides standards for diagnosing causes of PAH (i.e. lung disease, heart disease, pulmonary vascular obstructions, and collagen fiber disease)5. However, the underlying causes of PAH oftentimes may not be readily apparent, requiring physicians to consider all potential causes without the overt signs of underlying disease. This issue is further exacerbated by the limited knowledge surrounding risk factors. For example, PAH may be linked to HIV or substance abuse in some cases5. Additionally, biological sex plays a role in the diagnosis and treatment outcomes for PAH. Thus, research in the field of pulmonary hypertension aims to evaluate the correlation between certain risk factors and comprehend the underlying mechanisms that impact the progression of PAH.

Right Ventricular Structural Remodeling: Elastin and Collagen

In patients with pulmonary arterial hypertension (PAH), there is an increase in the pulmonary arterial afterload that causes the right ventricle (RV) to adapt. This remodeling occurs at multiple scales. Organ-level hemodynamic changes, myocardial thickening, and elastin and collagen fiber reorientation have been studied to better understand RV remodeling4.

The right ventricular free wall (RVFW) undergoes fibrosis which causes fiber reorientation in the extracellular matrix (ECM). The ECM is composed of complex collagen fiber networks, elastin fibers, and other connective tissues that are overlaid and have varying orientations throughout the tissue thickness. In healthy individuals, collagen makes up 4-9% of the RVFW area and plays a key role in passive RV mechanics4. Studies have shown that the mechanical contribution of the ECM increases as there is greater collagen recruitment which is directly correlated with RV stiffness. As PAH progresses, there is an increase in the RVFW collagen composition due to the heart’s prolonged exposure to high afterload pressures. This also causes the fibers to reorient and shift angles. Collagen fibers tend to run parallel to the myofibers which is a geometric property that results in anisotropic behavior at the tissue level4. Previous studies have shown that an increase in RV stiffness and remodeling in the longitudinal (apex-to-outflow) direction is associated with fiber reorientation from an endocardial and epicardial spiral to nearly homogeneous in the longitudinal direction5. Established techniques for analyzing RV fiber orientation include histological analysis, diffusion tensor MRI (DT-MRI), and second-harmonic generation (SHG) microscopy.

Planar Biaxial Testing

The biomechanical properties of soft tissue are an important indicator of the tissue’s health. For example, pulmonary arterial hypertension is associated with an increase in arterial stiffness, making it a valuable marker of the severity and progression of the disease, and subsequently correlated with the risk of negative cardiovascular events6. Planar biaxial testing devices are designed to measure the anisotropic properties of soft tissue samples, thus their properties in 2D space. The primary goal of the device is to develop a constitutive model to predict the mechanical behavior of the soft tissue sample. To this end, planar biaxial tests can induce a wide range of forces and shears on a sample, such as in-plane shear forces, and various strain rates and states. The anisotropic nature of soft tissue necessitates a device be able to test a soft tissue sample in multiple directions, thus in 2D space. Additionally, to simplify the analysis, the third dimension, the thickness of the tissue, is kept constant7.

There are a multitude of planar biaxial testing devices, but all entail attachment of a soft tissue sample onto the device and stretching it to induce the desired forces and shears8. The sample is typically mounted to the device using a gripping mechanism that are connected to the force actuators that stretch the sample9. The sample is submerged in phosphate-buffered saline (PBS) with a pH of 7.4, at room temperature, to mimic physiological or ex-vivo conditions8. Each sample is stretched only to a certain extent to prevent structural damage to the soft tissue10. Markers are inserted into the soft tissue to measure the deformation of the sample, with such measurements taken by a camera mounted above the sample8,10. During the experiment, tissue samples are preloaded and preconditioned, to ensure reproducible results10.

Second Harmonic Generation

Multi-photon microscopy is a powerful method for evaluating deep tissues and the ECM without visual artifacts or damage to the specimen. Second harmonic generation (SHG) involves a nonlinear optical effect where two photons of the same frequency interact with a nonlinear material and generate a photon that has twice the energy of the original photons. This phenomenon can only occur in media that do not have a center of symmetry (such as an anisotropic material) and can be used for tissues and protein arrays16.

SHG microscopy has been used to study the orientation and dispersion of myofibers and collagen fibers due to its high resolution which can provide information throughout the thickness of the tissue. It can be used to analyze the fiber and sheet orientation and angular fiber dispersion which are associated with cardiac diseases including PAH. Although other imaging modalities such as multi-photon microscopy are useful for 3D visualization, nonlinear techniques such as SHG allow for the labeling of proteins such as collagen, elastin, and myocardial fibers. By combining optical tissue clearing with SHG, one study increased the penetration depth achieved17. They used a picosecond laser source with an optical parametric oscillator at 880 nm integrated with a confocal microscope to visualize the collagen in the ventricular myocardium. By obtaining stacks of 2D images in the (x,y) plane, 3D projections can be reconstructed using image analysis to study the changes in fiber orientation post-biaxial tensile testing. However, there is currently no established method of performing biaxial testing concurrently with SHG imaging.

3D Printing

Fabrication of equipment remains costly when machining different materials. The use of 3D printing allows for inexpensive manufacturing of a variety of things. 3D printing has evolved into many different fields, especially the biomedical industry, as it can be used for testing devices as well as biological replacements11,12,13.

There are different options when it comes to 3D printing. The most common ones are accomplished by using a plastic polymer or a resin. The typical plastic polymer used is PLA, which when printed forms a hard rigid plastic. This is typically used for making structures, but can also have a rough exterior to it12,14. The limitations of this type of material being used are the lack of flexibility and the rough exterior that can have an increased impact on moving parts with increased friction. Another disadvantage is that printing multiple components takes much longer as there is only one nozzle where the plastic comes out. The other common type of material is resin, which is printed using a stereolithography printer. This material is more delicate, but the print is more precise and typically has much less tolerancing. The surface is much smoother16,14,15. The downside to this type of printing is that the material is toxic and must be cleaned properly to ensure that it is safe to touch.

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