Comparative multi-scale hierarchical structure of the tail, plantaris, and Achilles tendons in the rat

Abstract

Rodent tendons are widely used to study human pathologies such as tendinopathy and repair, and to address fundamental physiological questions about development, growth, and remodeling. However, how the gross morphology and multi-scale hierarchical structure of rat tendons, such as the tail, plantaris, and Achilles tendons, compare with that of human tendons are unknown. In addition, there remains disagreement about terminology and definitions. Specifically, the definitions of fascicle and fiber are often dependent on diameter sizes, not their characteristic features, and these definitions impair the ability to compare hierarchical structure across species, where the sizes of the fiber and fascicle may change with animal size and tendon function. Thus, the objective of the study was to select a single species that is commonly used for tendon research (rat) and tendons with varying mechanical functions (tail, plantaris, Achilles) to evaluate the hierarchical structure at multiple length scales using histology, SEM, and confocal imaging. With the exception of the specialized rat tail tendon, we confirmed that in rat tendons there are no fascicles and the fiber is the largest subunit. In addition, we provided a structurally based definition of a fiber as a bundle of collagen fibrils that is surrounded by elongated cells, and this definition was supported by both histologically processed and unprocessed samples. In all rat tendons studied, the fiber diameters were consistently between 10 and 50 μm, and this diameter range appears to be conserved across larger species. Specific recommendations were made highlighting the strengths and limitations of each rat tendon as a research model. Understanding the hierarchical structure of tendon can advance the design and interpretation of experiments and development of tissue-engineered constructs.

Keywords: fascicle; fiber; hierarchical structure; imaging; multi-scale.

Figure 1

Gross morphology of plantaris and Achilles tendons. (a) Plantaris (P) and Achilles (Ach) tendons are bound together in situ (dashed yellow line). (b) Plantaris muscle is posterior to Achilles muscle complex (dashed yellow line), and plantaris tendon is visible above Achilles tendon (solid yellow line). (c) By cutting connective tissue binding plantaris and Achilles tendons from the insertion, these two tendons can be completely separated (d). (e) Achilles tendon was further dissected and is a fusion of three tendons originating from soleus (Sol), lateral gastrocnemius (Lg), and medial gastrocnemius (Mg) muscles. (f) Sub‐tendons are tightly fused, and further separation of tendons is difficult without directly cutting the tendon. Scale bars: 2 mm. We created schematics of (g) posterior and (h) anterior views to show the complex structure of Achilles tendon.

Figure 2

Evaluation of fiber structure in longitudinal direction. (a‐c) Rat tail, plantaris, and Achilles tendons were cut in longitudinal (Long) direction, stained with PicroSirius Red, and viewed at low magnification. White boxes represent areas observed at (d‐f) high magnifications, and fibers are highlighted (white arrows). (g‐i) Longitudinal SEM shows fibers (green) with a similar diameter as observed in histology. No fascicle boundary was observed.

Figure 3

Evaluation of fiber structure in transverse direction. (a‐c) Rat tail, plantaris, and Achilles tendons were cut in the transverse direction, stained with H&E, and viewed at low magnification. Black boxes represent areas observed at (d‐f) high magnifications. The distance between cells are highlighted (blue arrow) for the rat tail tendon, and fibers in plantaris and Achilles tendons are outlined (blue). No fascicle boundary was observed.

Figure 4

Collagen alignment and cell morphology. (a‐c) Collagen alignment was viewed with SHG signals. Collagen was aligned at deep planes, away from the skin, for tail, plantaris, and Achilles tendons. (d‐f) Whereas the collagen alignment was not different between the superficial, close to the skin, and deep planes for the tail tendon, plantaris and Achilles tendons had an additional layer of a complex meshwork of collagen or peritenon. (g,h) In deep planes, elongated cells outlined collagen bundle (white arrows), which matched the sizes of fiber observed with histology. Thus, we define fiber as a bundle of collagen with diameter 10–50 μm outlined by elongated cells. No fascicle boundary was observed.

Figure 5

3D structure of tendon. (a‐c) 3D structure of tendon was rendered from a confocal imaging stack (50 μm deep in z‐direction) to show the overall cell morphology and collagen alignment. All tendons show that cells in the deep region (green) outline collagen (gray). For plantaris and Achilles, blood vessels and cells on peritenon (red) were observed, unlike rat tail tendon. (d‐f) Transverse view, x–z plane, without blood vessels further illustrates that elongated cells are inbetween adjacent fibers (yellow arrows) with a consistent diameter. (g‐i) Oblique view, x–y plane, shows a spacing between collagen (yellow arrows). The opacity of collagen was adjusted to show the 3D structure.

Figure 6

Schematic of hierarchical structure of rat tendons. Hierarchical structure of rat tail and plantaris and Achilles tendons were recreated based on the evidence from this study. Data incorporated in the schematic were adapted from Starborg et al. (2013), Szczesny & Elliott (2014b), and de Almeida Mdos et al. (2015).