Collagen fibril morphology and mechanical properties of the Achilles tendon in two inbred mouse strains

Abstract

The relationship between collagen fibril morphology and the functional behavior of tendon tissue has been investigated in numerous experimental studies. Several of these studies suggest that larger fibril radius is a primary determinant of higher tendon stiffness and strength; others have shown that factors apart from fibril radius (such as fibril-fibril interactions) may be critical to improved tendon strength. In the present study, we investigate these factors in two inbred mouse strains that are widely used in skeletal structure-function research: C57BL/6J (B6) and C3H/HeJ (C3H). The aim was to establish a quantitative baseline that will allow one to assess how regulation of tendon extracellular matrix architecture affects tensile mechanical properties. We specifically focused on collagen fibril structure and glycosaminoglycan (GAG) content--the two primary constituents of tendon by dry weight--and their potential functional interactions. For this purpose, Achilles tendons from both groups were tested to failure in tension. Tendon collagen morphology was analyzed from transmission electron microscopy images of tendon sections perpendicular to the longitudinal axis. Our results showed that the two inbred strains are macroscopically similar, but C3H mice have a higher elastic modulus (P < 0.05). Structurally, C3H mice showed a larger collagen fibril radius compared to B6 mice (96 +/- 7 nm and 80 +/- 10 nm respectively). Tendons from C3H mice also showed smaller specific fibril surface (0.015 +/- 0.001 nm nm(-2) vs. 0.017 +/- 0.003 nm nm(-2) in the B6 tendons, P < 0.05), and accordingly a lower concentration of GAGs (0.60 +/- 0.07 microg mg(-1) vs. 0.83 +/- 0.11 microg mg(-1), P < 0.05). As in other studies of tendon structure and function, larger collagen fibril radius appears to be associated with stiffer tendon, but this functional difference could also be attributed to reduced potential surface area exchange between fibrils and the surrounding proteoglycan-rich matrix, in which the hydrophilic GAG side chains may promote inter-fibril sliding. This study provides an architectural and functional baseline for a comparative murine model that can be used to investigate the genetic regulation of tendon biomechanics.

Fig. 1

Representative transmission electron micrograph for the two inbred strain, B6 and C3H, and its binarized reconstruction on the right. The binarized reconstruction is processed to calculate the fibril radius, the collagen area fraction, the interfibrillar distance, the specific fibril surface and the fibril contact area. The color bar indicates the pixel size of the fibril radius that is converted to nm. The black bar indicates 400 nm.

Fig. 2

Schematic representation of the morphological parameters measured with the automatic script. (A) Fibril radius, (B) collagen area fraction, (C) interfibrillar distance, (D) specific fibril surface, (E) fibril contact area.

Fig. 3

Cumulative distribution curves for B6 and C3H mice. B6 generally contain a majority of small fibrils, whereas C3H have a more uniformly distributed fibril radius. Histograms of the collagen fibril distribution for both groups. n is the total number of fibrils.

Fig. 4

Representative transmission electron micrograph of a longitudinal section of the Achilles tendon for (A) B6 and (B) C3H at a magnification of ×53 000. The black lines between the fibrils are the stained GAGs. (C) Means and standard deviations of sulfated GAG content in the two groups. *Statistically significant for P-value ≤ 0.05.