Characterizing the collagen fiber orientation in pericardial leaflets under mechanical loading conditions

Abstract

When implanted inside the body, bioprosthetic heart valve leaflets experience a variety of cyclic mechanical stresses such as shear stress due to blood flow when the valve is open, flexural stress due to cyclic opening and closure of the valve, and tensile stress when the valve is closed. These types of stress lead to a variety of failure modes. In either a natural valve leaflet or a processed pericardial tissue leaflet, collagen fibers reinforce the tissue and provide structural integrity such that the very thin leaflet can stand enormous loads related to cyclic pressure changes. The mechanical response of the leaflet tissue greatly depends on collagen fiber concentration, characteristics, and orientation. Thus, understating the microstructure of pericardial tissue and its response to dynamic loading is crucial for the development of more durable heart valve, and computational models to predict heart valves' behavior. In this work, we have characterized the 3D collagen fiber arrangement of bovine pericardial tissue leaflets in response to a variety of different loading conditions under Second-Harmonic Generation Microscopy. This real-time visualization method assists in better understanding of the effect of cyclic load on collagen fiber orientation in time and space.

FIGURE 1

The custom made biaxial testing machine. (A) A rectangular stage insert (a) with four chamfered corners was built to fit into the X–Y scanning stage (b). The platform geometry (c) was designed so that a pulley system (d) would allow force transfer from the grips (e), located on each side of the specimen, to the force gauges (f), located on the outskirts of the platform. A 10–32 screw (g) was considered between each grip and its corresponding force gauge to achieve tension control; (B) the whole view of the loading-imaging experimental system used in this study.

FIGURE 2

(a) Schematic representation of different loading configurations of the pericardial tissue loading-imaging experiment. The uniaxial longitudinal experiments were performed by applying the loads 1 and 3. The loads 2 and 4 were applied for uniaxial transverse experiments while all the loads were considered for biaxial experiment. The orientation of the fibers at the surface was considered the transverse direction in this experiment. Each load magnitude was 1.2 N and the tissue segment was 3 cm by 3 cm. The imaging was repeated at three centrally-located spots in each situation; (b) device stage insert during a sample device run. It shows how two grips were attached to a tissue segment to exert uniaxial tension on it.

FIGURE 3

Fourier analysis measures average fiber orientation. (a) A sample SHG collagen image excited at 900 nm with a Zeiss 510 Meta multiphoton microscopy; (b) and (c) Fourier transform images reveal mean fiber direction. These images are the natural logarithm of the FFT magnitudes (computed in Matlab with FFT2) with frequencies shifted to bring the zero-frequency to the center of the image. After segmenting the image by k-means clustering, linear regression was performed to determine the orientation of the long axis of the central bright region in each transform. The mean fiber direction is rotated 90° from that axis.

FIGURE 4

Collagen fiber distributions for the first sample sheet of bovine pericardium taken by loading-imaging technique described. They show the change in the orientation of collagen fibers for relaxed, longitudinal loading and unloading states every 10 µm for up to 60 µm depth. The unloading images were taken in two different steps; simultaneously after unloading and 10 min after unloading. As can be seen collagen fibers orientation change profoundly during loading condition and then reorient at the same position in their relaxed state after unloading. The angle of loading is 90° with respect to the conventional x axis.

FIGURE 5

Comparison of collagen fiber orientation in relaxed, loaded and unloaded (right after loading and 10 min after loading) states of the first bovine pericardial sheet. Uniaxial longitudinal loading state shows that fibers are almost perpendicular to force at the surface and in-line with the force in deeper layers.

FIGURE 6

Collagen fiber distributions for the first sample sheet of bovine pericardium taken by loading-imaging technique described. They show the change in the orientation of collagen fibers for relaxed, transverse loading and unloading states every 10 µm for up to 60 µm depth. The unloading images were taken in two different steps; simultaneously after unloading and 10 min after unloading. As can be seen collagen fibers orientation change differently than the longitudinal situation but they have the similar behavior after unloading. The angle of loading is 0° with respect to the conventional x axis.

FIGURE 7

Comparison of collagen fiber orientation in relaxed, loaded and unloaded (right after loading and 10 min after loading) states of the first bovine pericardial sheet. Uniaxial transverse loading state shows the same phenomena as the uniaxial longitudinal loading but with almost 90° angle shift in deeper layers.

FIGURE 8

Collagen fiber distributions for the first sample sheet of bovine pericardium taken by loading-imaging technique described. They show the change in the orientation of collagen fibers for relaxed, biaxial loading and unloading states every 10 µm for up to 60 µm depth. The unloading images were taken in two different steps; simultaneously after unloading and 10 min after unloading. As can be seen collagen fibers orientation is no longer in the direction of principal stresses while they show a similar behavior to the uniaxial ones after unloading.

FIGURE 9

Comparison of collagen fiber orientation in relaxed, loaded and unloaded (right after loading and 10 min after loading) states of the first bovine pericardial sheet. Biaxial loading state shows that fibers arrange almost at 60° angle in deeper layers while they are almost at 120° at the surface.

FIGURE 10

Comparison of collagen fiber orientation in relaxed, loaded and unloaded (right after loading and 10 min after loading) states of the second bovine pericardial sheet. Uniaxial longitudinal loading state shows that fibers are almost perpendicular to force at the surface and in-line with the force in deeper layers. The relaxed state shows quite different fiber orientation with respect to the first sample.

FIGURE 11

Comparison of collagen fiber orientation in relaxed, loaded and unloaded (right after loading and 10 min after loading) states of the second bovine pericardial sheet. Uniaxial transverse loading state shows that the fibers are in-line with the load at deeper layers.

FIGURE 12

Comparison of collagen fiber orientation in relaxed, loaded and unloaded (right after loading and 10 min after loading) states of the second bovine pericardial sheet. Biaxial loading state shows that fibers arrange almost at 50° angle in deeper layers while they are almost at 120° at the surface.

FIGURE 13

Comparison of collagen fiber orientation in relaxed, loaded and unloaded (right after loading and 10 min after loading) states of the third bovine pericardial sheet. Uniaxial longitudinal loading state shows that fibers are almost perpendicular to force at the surface and in-line with the force in deeper layers. The relaxed state shows quite different fiber orientation with respect to the first and second samples.

FIGURE 14

Comparison of collagen fiber orientation in relaxed, loaded and unloaded (right after loading and 10 min after loading) states of the third bovine pericardial sheet. Uniaxial transverse loading state shows that the fibers are in-line with the load at deeper layers and almost at 60° at the surface.

FIGURE 15

Comparison of collagen fiber orientation in relaxed, loaded and unloaded (right after loading and 10 min after loading) states of the third bovine pericardial sheet. Biaxial loading state shows that fibers arrange almost at 45° angle in deeper layers while they are almost at 130° at the surface.

FIGURE 16

Comparison of collagen fiber orientation in biaxially loaded vs. superposition of uniaxially loaded (longitudinal plus transverse) states of all bovine pericardial sheet samples. The graphs show that the biaxial behavior is totally different from uniaxial superposed data for all samples.