3-D ultrastructure and collagen composition of healthy and overloaded human tendon: evidence of tenocyte and matrix buckling

Abstract

Achilles tendinopathies display focal tissue thickening with pain and ultrasonography changes. Whilst complete rupture might be expected to induce changes in tissue organization and protein composition, little is known about the consequences of non-rupture-associated tendinopathies, especially with regards to changes in the content of collagen type I and III (the major collagens in tendon), and changes in tendon fibroblast (tenocyte) shape and organization of the extracellular matrix (ECM). To gain new insights, we took biopsies from the tendinopathic region and flanking healthy region of Achilles tendons of six individuals with clinically diagnosed tendinopathy who had no evidence of cholesterol, uric acid and amyloid accumulation. Biochemical analyses of collagen III/I ratio were performed on all six individuals, and electron microscope analysis using transmission electron microscopy and serial block face-scanning electron microscopy were made on two individuals. In the tendinopathic regions, compared with the flanking healthy tissue, we observed: (i) an increase in the ratio of collagen III : I proteins; (ii) buckling of the collagen fascicles in the ECM; (iii) buckling of tenocytes and their nuclei; and (iv) an increase in the ratio of small-diameter : large-diameter collagen fibrils. In summary, load-induced non-rupture tendinopathy in humans is associated with localized biochemical changes, a shift from large- to small-diameter fibrils, buckling of the tendon ECM, and buckling of the cells and their nuclei.

Keywords: 3View®; collagen; cross-links; fibers; fibrils; serial block face-scanning electron microscopy.

Figure 1

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of pepsin-solubilized collagens from healthy and tendinopathic sites in six individual patient tendons. The identities of the alpha chains of type I, III and V collagen are as indicated and were confirmed by mass spectroscopy.

Figure 2

Transmission electron microscopy. Typical electron microscope images of normal (A) and tendinopathic (B) tendon. Arrows indicate the axes of orientation, with x and y representing a plane at right angles (transverse) to the long axis of the tendon.

Figure 3

Fibril diameter distributions in healthy and tendinopathic regions. (A) The frequency distributions of collagen fibril diameters from the healthy regions and tendinopathic regions of tendon from two patients are shown. (B) The data are re-plotted to illustrate the distribution of large-diameter fibrils, which are most evident in healthy regions. P-values show significant differences between healthy and tendinopathic regions (Mann–Whitney U non-parametric tests).

Figure 4

Tenocytes in healthy tendon. SBF-SEM image of a tenocyte in a healthy region of tendon. The cell is surrounded by a well-organized ECM containing collagen fibrils. Arrows indicate long cellular processes that project deep into the ECM.

Figure 5

Disorganization of the ECM in tendinopathic regions. Tendinopathic tissue was examined by SBF-SEM and subsequently analyzed by IMOD. (A) Section 450 (of 740 × 100-nm-thick sections) showing cross-sections of two cells that are surrounded by curvilinear bundles of collagen fibrils. Arrow indicates electron-lucent regions between cells and the surrounding ECM. (B) Segmentation of some of the objects shown in (A). Blue, nuclear membrane. Yellow and orange, plasma membranes. Purple, green and yellow, collagen fibril bundles. Two cells are segmented (one colored orange, the other colored yellow). The nucleus of the cell bounded in orange is not visible because it is located at a different z-height in relation to the cell bounded in yellow.

Figure 6

Automated segmentation in IMOD identifies changes in the organization in normal and tendinopathic regions of the same tendon. (A) Healthy tendon shows near-parallel alignment of collagen fibrils (oriented north–south). (B) Tendinopathic tendon contains disorganized ECM. Arrows show orientation and are 10-μm scale bars.

Figure 7

Three-dimensional organization of tenocytes in healthy and tendinopathic regions. (A) Healthy tendon. Nuclei are shown in different hues of blue and are aligned parallel to the tendon long axis. (B) Tendinopathic region of tendon. Nuclei are distributed almost randomly in three-dimensions. The images are frame shots of Videos S3 and S4. An electron microscope image is shown superimposed on the 3D reconstructions. The sizes of the images are 40 × 40 μm (x–y-axes). The scale bars on the z-axis (i.e. parallel to the tendon long axis) are 10 μm.

Figure 8

Buckling and slippage of cells in tendinopathic regions. (A) Three-dimensional reconstruction showing two cells in healthy tendon. The large cell in the center of the view is undergoing mitosis and is aligned parallel to the tendon long axis. Both cells are stacked one-on-top of the other. (B) Three-dimensional reconstruction showing two cells in tendinopathic tendon. The cells are aligned side-by-side, which was not observed in healthy tendon. (C) Schematic representation of cell slippage to explain the change in cell orientation from head-to-tail to side-by-side alignment.