Region- and layer-specific investigations of the human menisci using SHG imaging and biaxial testing

FIGURE 1. Classification of the meniscus tissue into three regions and two layers (A) The top view of the lateral meniscus of the left knee with identification of the posterior, mid-body, and anterior regions. (B) The location of the samples on the cross-sectional view.

FIGURE 2. Sample preparation and mounting protocol for biaxial tension testing (A) The sample is placed on a 3D-printed template. The sample is attached with barbless hooks and sutures, marked using tissue marking dye. (B) Custom-built biaxial tensile test set-up. The sample is submerged in PBS. (C) The sample after optical tissue clearing.

FIGURE 3. Postprocessing of the obtained MPM image stacks (A) The recorded SHG signals through the thickness of the tissue in 2 μm intervals with each image in the stack analyzed to extract the planar orientation distribution. (B) The computed orientation distributions of all images of the MPM stack as an intensity plot with 1 representing the peak of collagen intensity and 0 representing low collagen intensity. (C) The corresponding planar orientation distribution of an individual slice of the MPM stack along with its relative position in the 3D intensity plot.

FIGURE 4. Computed Cauchy stress-strain curves for the meniscus samples (LM1 and MM1) obtained from the biaxial testing protocol. The top and bottom rows of subplots represent the samples from the top and bottom samples, respectively. Column-wise, they represent the anterior, mid-body, and posterior regions.

FIGURE 5. Depth profile of collagen fiber intensity of samples obtained from the lateral meniscus. The images recorded on the entire stack were analyzed individually and compiled together in Matlab to produce an intensity plot representative of the transmural variation in collagen fiber morphology. The top and bottom rows of subplots represent the top and bottom samples, respectively. Column-wise, they represent the anterior, mid-body, and posterior regions.

FIGURE 6. The orientation distribution computed on each image slice of individual image stacks was averaged through the thickness to obtain the representative collagen orientation distribution of the sample. Here, the computed orientation distribution for samples excised from lateral menisci is shown. The top and bottom profiles represent the samples from the top and bottom samples, respectively. Column-wise, they represent the anterior, mid-body, and posterior regions.

FIGURE 7. Depth profile of collagen fiber intensity of samples obtained from the medial meniscus. The images recorded on the entire stack were analyzed individually and compiled together in Matlab to produce an intensity plot representative of the transmural variation in collagen fiber morphology. The top and bottom rows of subplots represent the top and bottom samples, respectively. Column-wise, they represent the anterior, mid-body, and posterior regions.

FIGURE 8. The orientation distribution computed on each image slice of individual image stacks was averaged through the thickness to obtain the representative collagen orientation distribution of the sample. Here, the computed orientation distribution for samples excised from medial menisci is shown. The top and bottom profiles represent the samples from the top and bottom samples, respectively. Column-wise, they represent the anterior, mid-body, and posterior regions.

FIGURE 9. Cauchy stress versus strain for a sample in the radial (red curve) and circumferential direction (blue curve). The experimental data (solid curve) is well represented by the model (dotted curve) with r 2 of 0.94. On the final loading regime of the red curve, the computation of linear elastic modulus is illustrated. Most of the tissue stiffness in this loading region is assumed to be contributed by fibers.

FIGURE 10. Computed elastic modulus of a human lateral meniscus in the final loading regime: top layer (left), and bottom layer (right). The anisotropic nature of the material is shown with the circumferential stiffness represented in blue and the radial stiffness represented in red. Within each layer, the anterior, middle, and posterior regions are presented to see the regional variations. * indicates statistical significance from a one-way ANOVA test with p-value < 0.05.

FIGURE 11. Computed elastic modulus of a human medial meniscus in the final loading regime: top layer (left), and bottom layer (right). The anisotropic nature of the material is shown with the circumferential stiffness represented in blue and the radial stiffness represented in red. Within each layer, the anterior, middle, and posterior regions are presented to see the regional variations. * indicates statistical significance from a one-way ANOVA test with p-value < 0.05.

FIGURE 12. Computed biaxial elastic modulus of all meniscus samples showing regional and depth-wise variation. The circumferential and radial moduli are shown as dotted and solid lines respectively. Regions anterior (red), middle (blue), and posterior (black) are presented here.