Demonstration of Quantitative Fabric Analysis of Tendon Collagen using Two-Dimensional Polarized Light Microscopy

doi:10.1016/S0934-8832(11)80227-1

Matrix

Volume 11, Issue 1, February 1991, Pages 56-62

https://doi.org/10.1016/S0934-8832(11)80227-1 Get rights and content

Abstract

The extensional behavior of tendon is closely linked to the directional organization of the collagen fibers in the tendon. We sought to demonstrate that quantitative information about this organization is accessible through the application of polarized light microscopy. Ten tendons (2 canine Achilles and 8 rat tail tendons) were sectioned in a longitudinal plane and stained with either hematoxylin and eosin or toluidine blue. One Achilles tendon was fixed without tension on the fibers, thus leaving the fibers in a wavy state, and the other was fixed while strained with longitudinal tension straightening the fibers. One hundred measurements of collagen fiber orientation were made on a 0.7 mm² area from each tendon. Recorded orientation angles were plotted on circular histograms and analyzed using circular statistics. The strained fibers revealed a sharply peaked distribution, in contrast the unstrained fibers had a bimodal orientation distribution. The tail tendons were fixed at strains from 0–3%. Analysis of the orientation distributions revealed increasing alignment of the collagen as strain increased. These data, while matching the qualitative descriptions of the fibers, also provide quantitative assessment of organization enabling comparison among tissues of varied physical circumstances and function. This method of quantitative orientation measurement has wide application for the many birefringent tissues such as bone, smooth and striated muscle and elastin.

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The region of interest was set as a standardized field (3 × 1 mm) at the bone-tendon junction (Fig. 1, D, b). The spatially aligned collagen fibers were assessed on HE-stained paraffin sections using polarization microscopy.36 Polarized light directed at oriented collagen fibers in tissue sections is diffracted and shines brightly against a dark background.19
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Treatment with OCP made collagen fibers and the Sharpey fibers, constituted by type I and type III collagens, increase at the tendon-to-bone insertion. It might be beneficial for the healing of rotator cuff tendon to bone.
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As such, polarized light has been used to assess microstructural tissue changes as a means of estimating the onset of tissue injury. Polarized light has been used to quantify the collagen orientation and alignment in a variety of different tissues including the human and canine Achilles tendon, rat tail, human and rat cervical facet ligament, and the supraspinatus muscle (Dickey et al., 1998; Quinn and Winkelstein, 2008; Quinn et al., 2010; Thomopoulos et al., 2003; Whittaker and Canham 1991). This technique takes advantage of collagen's natural birefringence to estimate the fiber alignment of such tissues (Tower and Tranquillo, 2001).
Studies implicate the cervical facet joint and its capsule as a primary anatomical site of injury during whiplash exposures to the neck. Although the facet joint is known to undergo stretch as the superior vertebra is retracted relative to the inferior vertebra during the whiplash kinematic, the response of the facet capsular ligament and its microstructure during failure in joint retraction is unknown. Polarized light imaging and vector correlation analysis were used to measure the collagen fiber alignment in the human capsular ligament, together with traditional mechanical metrics, during joint retraction sufficient to induce ligament failure. Anomalous fiber realignment occurs at 2.95±1.66 mm of displacement, which is not different from the displacement when the ligament first yields (2.77±1.55 mm), but is significantly lower (p=0.016) than the displacement at tissue failure (5.40±1.65 mm). The maximum principal strain at the first detection of anomalous fiber realignment (0.66±0.39) also is significantly lower (p=0.046) than the strain at failure (1.39±0.64), but is not different from the strains at yield or partial failure. The onset of collagen fiber realignment determined in this study corresponds to the ligament's yielding and supports assertions that the facet capsule can undergo tissue injury during joint retraction. Further, such microstructural responses may indicate tissue damage in the absence of rupture.

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^a: Peter Whittaker, Ph. D., The Heart Institute, Hospital of the Good Samaritan, 616 S. Witmer Street, Los Angeles, CA 90017, USA.

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Demonstration of Quantitative Fabric Analysis of Tendon Collagen using Two-Dimensional Polarized Light Microscopy

Abstract

Int. J. Biol. Macromol.

Am. Heart J.

Matrix

Collagen organisation in the interspinous ligament and its relationship to tissue function

J. Anat.

Circular Statistics in Biology

The microscopical investigation of biological materials with polarized light

The tunica muscularis of human brain arteries

Neurol. Res.

Fibre-buckling in composite systems

J. Materials Sci.