Elastin governs the mechanical response of medial collateral ligament under shear and transverse tensile loading

Abstract

Elastin is a highly extensible structural protein network that provides near-elastic resistance to deformation in biological tissues. In ligament, elastin is localized between and along the collagen fibers and fascicles. When ligament is stretched along the primary collagen axis, elastin supports a relatively high percentage of load. We hypothesized that elastin may also provide significant load support under elongation transverse to the primary collagen axis and shear along the collagen axis. Quasi-static transverse tensile and shear material tests were performed to quantify the mechanical contributions of elastin during deformation of porcine medial collateral ligament. Dose response studies were conducted to determine the level of elastase enzymatic degradation required to produce a maximal change in the mechanical response. Maximal changes in peak stress occurred after 3h of treatment with 2U/ml porcine pancreatic elastase. Elastin degradation resulted in a 60-70% reduction in peak stress and a 2-3× reduction in modulus for both test protocols. These results demonstrate that elastin provides significant resistance to elongation transverse to the collagen axis and shear along the collagen axis while only constituting 4% of the tissue dry weight. The magnitudes of the elastin contribution to peak transverse and shear stress were approximately 0.03 MPa, as compared to 2 MPa for axial tensile tests, suggesting that elastin provides a highly anisotropic contribution to the mechanical response of ligament and is the dominant structural protein resisting transverse and shear deformation of the tissue.

Keywords: Elastase; Elastin; Ligament; Shear; Transverse tensile.

Figure 1

Schematic of specimen harvest locations in porcine MCL. Two neighboring rectangular specimens were harvested at the midpoint of the ligament (punch footprint shown in red). A representative transverse tensile and shear specimen (blue) are shown as the area between the clamps with respect to the overall punch dimensions. Red arrows denote the axes of deformation relative to the collagen fibers. The shear specimen height was reduced to ensure the area between the clamps was nearly square, allowing for adequate tissue to be gripped in the clamps. Note that within a ligament both specimens were harvested for the same test protocol. Shear and transverse tensile are shown here in the same ligament for illustration only.

Figure 2

Elastin structure and degradation products. A: Elastin recoils due to hydrophobic and entropic forces, but extends under applied force (F). Desmosine and (iso)desmosine crosslinks resist network deformation. B: Elastase degrades elastin via cleavage of tropoelastin, leaving fragments with crosslinks intact.

Figure 3

Dose-response curves for transverse tensile tests. A: Peak stress decreased rapidly and fit an exponential decay function as the concentration of elastase increased (3 hr treatment for all specimens). The trend stabilized beyond 2 U/ml elastase. (*) significant difference with respect to control (0 U/ml). B: Peak stress decreased rapidly and fit an exponential decay function for both control and elastase treated pairs (2 U/ml elastase for treated specimens). Decay in the control samples is representative of tissue swelling in the buffer, which stabilized after 1 hr of treatment. The difference between control and treated samples also stabilized above 1 hr treatment. (*) significant difference between matched pairs of control and treated samples.

Figure 4

The stress-strain response of transverse tensile (A) and simple shear (B) specimens can be described by exponential growth curves. In both tests, stress and stiffness of the tissue significantly decreased after elastase degraded the elastin (*).

Figure 5

Complete quasi-static material characterization of control and elastase digested porcine MCL in (A) longitudinal tensile, (B) transverse tensile and (C) simple shear deformations. Note that (B) and (C) are on a stress scale two orders of magnitude smaller than (A), highlighting the anisotropic nature of the tissue construction. While elastin is the primary structural protein that supports nearly 70% of transverse and shear stress, the relative magnitude of the contribution is minute in comparison to that in the longitudinal deformation. Figure 5A [5] reproduced with permission (Wiley #3474980233721).

Figure 6

Multiphoton laser scanning microscopy images of porcine MCL. A: Collagen second harmonic generation signal collected at 860 nm excitation, emission filtered to 420-460 nm. Arrow denotes collagen axis. B: Elastin autofluorescence signal collected at 800 nm excitation, emission filtered to 495-540 nm. Elastin is localized between and along neighboring collagen fibers. C: Image of the elastin autofluorescence signal in tissue treated with elastase. Note the granulated appearance of the elastin and the lack of elastin interweaving the collagen.

Figure 7

Elastase activity assay for control and inhibited assays. A concentration of 1 U/ml of elastase inhibitor was found to quench the equivalent activity of 2 U/ml elastase as used in the mechanical tests.

Figure 8

SDS-PAGE of soluble collagen in the presence of enzymes. Elastase and inhibited elastase had no appreciable effect on collagen migration bands. Collagenase significantly degraded collagen as exhibited by the lack of collagen banding in the collagenase lanes.