The evolution of collagen fiber orientation in engineered cardiovascular tissues visualized by diffusion tensor imaging

Abstract

The collagen architecture is the major determinant of the function and mechanical behavior of cardiovascular tissues. In order to engineer a functional and load-bearing cardiovascular tissue with a structure that mimics the native tissue to meet in vivo mechanical demands, a complete understanding of the collagen orientation mechanism is required. Several methods have been used to visualize collagen architecture in tissue-engineered (TE) constructs, but they either have a limited imaging depth or have a complicated set up. In this study, Diffusion Tensor Imaging (DTI) is explored as a fast and reliable method to visualize collagen arrangement, and Confocal Laser Scanning Microscopy (CLSM) was used as a validation technique. Uniaxially constrained TE strips were cultured for 2 days, 10 days, 3 and 6 weeks to investigate the evolution of the collagen orientation with time. Moreover, a comparison of the collagen orientation in high and low aspect ratio (length/width) TE constructs was made with both methods. Both methods showed similar fiber orientation in TE constructs. Collagen fibers in the high aspect ratio samples were mostly aligned in the constrained direction, while the collagen fibers in low aspect ratio strips were mainly oriented in the oblique direction. The orientation changed to the oblique direction by extending culture time and could also be visualized. DTI captured the collagen orientation differences between low and high aspect ratio samples and with time. Therefore, it can be used as a fast, non-destructive and reliable tool to study the evolution of the collagen orientation in TE constructs.

Fig 1

(A) Schematic drawing of the polycarbonate frame, (B) low (size: 25×5×1 mm) and (C) high (size: 25×3×1 mm) aspect ratio TE strips glued to the frame just after seeding.

Fig 2. Geometric representation of the diffusion tensors, showing isotropic (A) and anisotropic (B) diffusion.

Fig 3. Artificial fibers with isotropic (A) and anisotropic (D) alignment, their corresponding histograms (B, E), and dispersity as ellipses (C, F).

Isotropic fibers are shown as a circle and anisotropic fibers as a line.

Fig 4. Macroscopic images of high aspect ratio TE strips cultured for 10 days (A), 3 weeks (C) and 6 weeks (E) and their corresponding CLSM images (B, D, F).

(Strip size: 25×3×1 mm) collagen fiber alignment changed to the constrained direction with time and changed to the oblique direction in regions where compaction is more pronounced. Scale bars represent 1 mm.

Fig 5. Macroscopic images of low (A) and high (C) ratio rectangular TE strips cultured for 3 weeks and corresponding CLSM images of collagen fibers (B, D).

The scale of alignment is shown by ellipses for each tile. Collagen fibers aligned in the oblique direction in low aspect ratio strips after 3 weeks, while they were aligned in the constrained direction in high aspect ratio strips. Scale bars represent 1 mm.

Fig 6. Arrays of ellipsoids obtained from the diffusion tensor of DTI scan in a single slice of middle part (10×3 mm) of TE samples after 2 days (A), 3 weeks (B) and 6 weeks of culture (C).

Ellipsoids show the orientation and magnitude of the diffusion in each voxel. Fibers were randomly oriented after 2 days, but they become aligned in the constrained direction after 3 weeks and in the oblique direction in more compacted regions after 6 weeks.

Fig 7. T₂-weighted DTI images of low (A) and high (D) aspect ratio TE strips after 3 weeks of culture.

Fiber tracts represent the fibrous structure in the engineered constructs with low (top view (B), side view (C)) and high (top view (D), side view (F)) aspect ratio. Fibers are color-coded based on their directions: red (x direction), green (y direction) and blue (z direction). Collagen fibers are mostly aligned in the constrained direction in high aspect ratio strips and in the oblique direction in low aspect ratio strips. Strip size in image (A-C): 10×5×1 mm, and in image (D-F): 10×3×1 mm.

Fig 8. The mean angle and dispersity of fiber alignment of low aspect ratio and high aspect ratio constructs after 3 weeks of culture (A,C) and high aspect ratio after 3 and 6 weeks of culture (B,D).

The mean angle of high aspect ratio group compared to low aspect ratio group and 6 weeks group compared to 3 weeks group was significantly changed. However, the dispersity was not significantly different between any of those groups. There was no significant difference between DTI and CLSM within any group. Each bar represents the mean ± SD (* represents p<0.05).

Fig 9. Hematoxylin and eosin staining of high aspect TE strips cultured for 2 days (A), 10 days (B), 3 weeks (C) and 6 weeks (D).

The scale bar represents 500 μm. By extending culture time, TE strips became more compacted. Dashed rectangles represent the original shape of the strips.

Fig 10. Hematoxylin and eosin staining of low (A) and high (C) aspect ratio TE sample after 3 weeks of culture.

Both samples were compacted after 3 weeks. The scale bar represents 500 μm.

Fig 11. Schematic representation of collagen orientation evolution with time in low (A-C) and high (D-F) rectangular TE strips.

The collagen orientation changes by culture time. First, the fibers were randomly oriented. By extending culture time, fibers aligned in the constrained direction in the high aspect ratio strips and in the oblique direction in low aspect ratio strips. By culturing for a longer time, the fibers aligned in the oblique direction in both configurations.