Figure 1.
The methodology and devices for measuring the sectional areas of specimens.
(a) Bone-ligament-bone samples (B) were fixed by clamps (D) with their longitudinal axes parallel to the direction of the motion unit (C). The contour profiles of the mid-substance specimen between 0–180 degrees and 180–360 degrees were measured by the two opposite sensors (A), respectively. And the whole profile could be obtained by combining the two separate profiles. (b) With the two sensors moving along the direction of the motion unit, two whole profile measurements (i.e., profile 1 & 2) were acquired at the cross sections that were 1 mm far from the mid-position. The mid-position was located at the central section between the femoral and tibial insertions. (c) An average sectional area for each mid-substance specimen was calculated by the two whole contour profiles.
Figure 2.
Experimental apparatus used to measure the mechanical behavior of the mid-substance specimen of rabbit MCL.
The tensile load was applied by an actuator (A), and recorded by a load cell (B) in the MTS testing machine. Two clamps (D) were adjusted to hold the bone-MCL-bone sample (E) vertically to ensure that the tensile load was applied along the longitudinal axis of the mid-substance specimen of the ligament. All the tests were performed in a bath chamber (C) at physiological temperature (controlled by a temperature sensor (F)) and humidity. The displacements of ink speckles on the mid-substance specimen were measured by a camera (G) and used to calculate the tensile strain by a custom-written MATLAB script.
Figure 3.
Example bilinear curvefitting of stress-strain tensile experimental data.
The arrow indicates the transition point of the fitting line from the toe-region to the linear region.
Figure 4.
A process flow diagram for differentiating the collagen contents in the histological slice of the mid-substance specimen.
Two slices were sectioned at the upper and lower boundaries of each mid-substance specimen, corresponding to the locations that 1 mm far from the mid-position of the ligament, respectively (shown as ‘slice 1 & 2’ in Figure 1). Each slice was firstly stained with 0.1% picrosirius red solutions, and imaged under polarized light (a). Then, the dark background of the image was removed for further image processing (b). The color results of all the pixels in each slice were obtained (c): the type I and type III collagen corresponded to the pixels with red-yellow color (hue values of 345–75 degrees) and greenish color (hue values of 75–165 degrees), respectively. Average collagen ratios were calculated from the collagen content values in the two slices for characterizing each mid-substance specimen. In addition, the distributions of types I and III collagen in the specimen slice were determined by the hue values of the pixels (d).
Figure 5.
Comparisons of the ratio of type I collagen (a), the ratio of type III collagen (b), and the Young's modulus in the linear region of the stress-strain curve (c) between the different age groups.
During the maturation of ligaments (i.e., from the 1.5-month-old group to the 12- to 13-month-old group), the ratio of type I collagen increased while the ratio of type III collagen decreased. Significant differences were found in the ratio of type I collagen between the 1.5-month-old group and the 6- to 7-month-old group, between the 1.5-month-old group and the 12- to 13-month-old group, and between the 4- to 5-month-old group and the 12- to 13-month-old group (P<0.05). The ratio of type III collagen in the 1.5-month-old group was significantly higher than that in the other groups (P<0.05). The Young's modulus in the linear region changed significantly between the 4- to 5-month-old group and the 12- to 13-month-old group as well as between the 1.5-month-old group and the other three groups (P<0.05). Mean±SD.
Table 1.
The age and body mass of rabbit, and the cross sectional area, collagen contents (ratio of type I collagen, , and ratio of type III collagen,
) and mechanical property (Young's modulus in the linear region of the stress-strain curve
) of the mid-substance specimens of MCL.