Effect of preconditioning and stress relaxation on local collagen fiber re-alignment: inhomogeneous properties of rat supraspinatus tendon

Abstract

Repeatedly and consistently measuring the mechanical properties of tendon is important but presents a challenge. Preconditioning can provide tendons with a consistent loading history to make comparisons between groups from mechanical testing experiments. However, the specific mechanisms occurring during preconditioning are unknown. Previous studies have suggested that microstructural changes, such as collagen fiber re-alignment, may be a result of preconditioning. Local collagen fiber re-alignment is quantified throughout tensile mechanical testing using a testing system integrated with a polarized light setup, consisting of a backlight, 90 deg-offset rotating polarizer sheets on each side of the test sample, and a digital camera, in a rat supraspinatus tendon model, and corresponding mechanical properties are measured. Local circular variance values are compared throughout the mechanical test to determine if and where collagen fiber re-alignment occurred. The inhomogeneity of the tendon is examined by comparing local circular variance values, optical moduli and optical transition strain values. Although the largest amount of collagen fiber re-alignment was found during preconditioning, significant re-alignment was also demonstrated in the toe and linear regions of the mechanical test. No significant changes in re-alignment were seen during stress relaxation. The insertion site of the supraspinatus tendon demonstrated a lower linear modulus and a more disorganized collagen fiber distribution throughout all mechanical testing points compared to the tendon midsubstance. This study identified a correlation between collagen fiber re-alignment and preconditioning and suggests that collagen fiber re-alignment may be a potential mechanism of preconditioning and merits further investigation. In particular, the conditions necessary for collagen fibers to re-orient away from the direction of loading and the dependency of collagen reorganization on its initial distribution must be examined.

Fig. 1

Angled side-view of the tendon tensile testing setup showing polarized light and imaging system: light source, rotating cross-polarized sheets, stepper motors, and camera

Fig. 2

Load-time graph of a representative sample undergoing the mechanical testing protocol. Fourteen-image alignment maps were taken for alignment analysis at (1) before preconditioning, (2) after preconditioning, (3) after preconditioning following a 300 s hold, (4) after the SR displacement, (5) during SR, (6) after SR following a return to zero displacement, (7) at the transition strain, and (8) at a point in the linear-region.

Fig. 3

Circular variance (VAR) values demonstrate increasing alignment (decreased VAR) during preconditioning, the displacement for SR in the toe and linear regions for midsubstance and insertion site. A decrease in alignment (increased VAR) was noted following the return to zero displacement for both locations. (*P < 0.0125, **P < 0.0025, ***P < 0.00025 = sig.).

Fig. 4

Linear modulus values obtained from the linear region of the optical stress-strain curve. Insertion site linear modulus is lower than the midsubstance indicating inhomogeneous mechanical properties of the rat SST. (***P < 0.00025 = sig.).

Fig. 5

Local optical strain values required to reach the transition point (intersection of toe and linear regions of the optical stress-strain curve) is shown for both locations. A higher local, optical strain is necessary for the insertion site of the tendon to transition to the linear region than for the tendon midsubstance. (***P < 0.00025 = sig.).

Fig. 6

Midsubstance histograms for a representative sample show increasing alignment during preconditioning, during the displacement for the stress relaxation test, followed by a decrease in alignment after returning to zero-displacement and increases in toe-region (comparing return to zero and transition) and linear-region (comparing transition and linear-region). (*P < 0.0125, **P < 0.0025, ***P < 0.00025 = sig.).