Structure and collagen crimp patterns of functionally distinct equine tendons, revealed by quantitative polarised light microscopy (qPLM)

Abstract

Structure-function relationships in tendons are directly influenced by the arrangement of collagen fibres. However, the details of such arrangements in functionally distinct tendons remain obscure. This study demonstrates the use of quantitative polarised light microscopy (qPLM) to identify structural differences in two major tendon compartments at the mesoscale: fascicles and interfascicular matrix (IFM). It contrasts functionally distinct positional and energy storing tendons, and considers changes with age. Of particular note, the technique facilitates the analysis of crimp parameters, in which cutting direction artefact can be accounted for and eliminated, enabling the first detailed analysis of crimp parameters across functionally distinct tendons. IFM shows lower birefringence (0.0013 ± 0.0001 [-]), as compared to fascicles (0.0044 ± 0.0005 [-]), indicating that the volume fraction of fibres must be substantially lower in the IFM. Interestingly, no evidence of distinct fibre directional dispersions between equine energy storing superficial digital flexor tendons (SDFTs) and positional common digital extensor tendons (CDETs) were noted, suggesting either more subtle structural differences between tendon types or changes focused in the non-collagenous components. By contrast, collagen crimp characteristics are strongly tendon type specific, indicating crimp specialisation is crucial in the respective mechanical function. SDFTs showed much finer crimp (21.1 ± 5.5 µm) than positional CDETs (135.4 ± 20.1 µm). Further, tendon crimp was finer in injured tendon, as compared to its healthy equivalents. Crimp angle differed strongly between tendon types as well, with average of 6.5 ± 1.4° in SDFTs and 13.1 ± 2.0° in CDETs, highlighting a substantially tighter crimp in the SDFT, likely contributing to its effective recoil capacity.

Statement of significance: This is the first study to quantify birefringence in fascicles and interfascicular matrix of functionally distinct energy storing and positional tendons. It adopts a novel method - quantitative polarised light microscopy (qPLM) to measure collagen crimp angle, avoiding artefacts related to the direction of histological sectioning, and provides the first direct comparison of crimp characteristics of functionally distinct tendons of various ages. A comparison of matched picrosirius red stained and unstained tendons sections identified non-homogenous staining effects, and leads us to recommend that only unstained sections are analysed in the quantitative manner. qPLM is successfully used to assess birefringence in soft tissue sections, offering a promising tool for investigating the structural arrangements of fibres in (soft) tissues and other composite materials.

Keywords: Birefringence; Collagen crimp; Fascicles; Interfascicular matrix (endotenon); Quantitative polarised light microscopy (qPLM); Tendon.

Graphical abstract

Fig. 1

Schematic of an equine superficial digital flexor tendon (SDFT) with an unstained histological section showing the fascicles and the interfascicular matrix (IFM) imaged using quantitative polarised light microscopy (qPLM). The two phases of the tendon mesostructure are clearly differentiated.

Fig. 2

(a) Schematic of the quantitative polarised light microscopy (qPLM) system used in the study. The qPLM technique outputs three matching images: a grey scale intensity image I/I₀ that can be used to distinguish tendon zones (fascicles and the interfascicular matrix) (b), a false-coloured image of the phase shift |sin δ| that is directly related to tendon birefringence which in turn can be related to the directional dispersion of collagen fibres in a tissue (c) and a false-coloured slow axis orientation image φ (d) intensifying the visibility of collagen crimp and facilitating fascicle crimp length measurements. Moreover the combination of c) and d) enables collagen crimp angle measurements without the sectioning artefact resulting from the unknown relationship between the sectioning and crimp propagation planes (details in 2.3, 2.4). The typical images from the three channels shown above were taken from an unstained SDFT section of a young horse (b–d). Scale bars = 100 µm.

Fig. 3

Crimp characteristics evaluated in slow axis images: a) an unstained section of a young CDET, with a black line representing a line plot of slow axis angles superimposed on the image, b) definition of collagen crimp length L and crimp angle θ_crimp, c–d) thresholding procedure selecting slopes of collagen crimp travelling to and away from a crimp peak. Scale bars = 100 µm.

Fig. 4

Comparison of matched unstained (a, c) and picrosirius red stained (b, d) sections of a superficial digital flexor tendon, SDFT (a, b) and a common extensor tendon, CDET (c, d). The false-coloured images encode the phase shift |sin δ| which is proportional to birefringence (see Eq. (1). Higher birefringence is observed in stained sections (b and d), as compared to the unstained sections (a and c). Scale bars = 100 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Slow axis orientation images showing crimp length (L) in superficial digital flexor tendon, SDFT (a) and common digital extensor tendon, CDET (b) tendons, as visualised with the false coloured orientation angle of the slow axis images. Notice the crimp length: L is much larger in CDET than in SDFT tendons. Scale bars = 100 µm.

Fig. 6

Unstained sections of injured superficial digital flexor tendon, SDFT: a) the false coloured image depicts the inhomogeneity of birefringence (white arrow) within a fascicle from an injured tendon, b) the slow axis orientation image depicts the difference in crimp length L on two sides of the interfascicular matrix (IFM). Scale bars = 100 µm.

Fig. 7

Crimp parameters (length and angle) related to the mechanical properties of fascicles: a) elastic modulus and b) failure strain. The data represents mean values (with bars representing standard deviations) of eight tendons (of 12 tested with qPLM) for which a matching experimental data has been acquired in a study by Thorpe et al. . Dots represent 2D projections of points into the x-y and y-z planes.