Concerted and adaptive alignment of decorin dermatan sulfate filaments in the graded organization of collagen fibrils in the equine superficial digital flexor tendon

Abstract

The equine superficial digital flexor tendon (SDFT) has a graded distribution of collagen fibril diameters, with predominantly small-diameter fibrils in the region of the myotendinous junction (MTJ), a gradual increase in large-diameter fibrils toward the osteotendinous junction (OTJ), and a mixture of small- and large-diameter fibrils in the middle metacarpal (MM) region. In this study, we investigated the ultrastructure of the SDFT, to correlate the spatial relationship of the collagen fibrils with the graded distribution. The surface-to-surface distances of pairs of fibrils were found to be almost constant over the entire tendon. However, the center-to-center distances varied according to fibril diameter. Decorin is the predominant proteoglycan in normal mature tendons, and has one dermatan sulfate (DS) or chondroitin sulfate (CS) filament as a side chain which is associated with the surfaces of the collagen fibrils via its core protein. We identified a coordinated arrangement of decorin DS filaments in the equine SDFT. The sizes of the decorin DS filaments detected by Cupromeronic blue staining showed a unique regional variation; they were shortest in the MM region and longer in the MTJ and OTJ regions, and a considerable number of filaments were arranged obliquely to adjacent collagen fibrils in the MTJ region. This regional variation of the filaments may be an adaptation to lubricate the interfibrillar space in response to local mechanical requirements. The results of this study suggest that the MTJ region, which receives the muscular contractile force first, acts as a buffer for mechanical forces in the equine SDFT.

Fig. 1

Schematic representation of the measurements of the center-to-center and surface-to-surface distances between collagen fibrils. The distances are defined according to Kuwaba et al. (2001). A collagen fibril (dark gray) surrounded by five or six adjacent fibrils (light gray) was randomly selected. The distance between the central fibril and each adjacent fibril was measured: 1, center-to-center distance; 2, surface-to-surface distance.

Fig. 2

Western blot analysis of decorin extracted from three regions of the superficial digital flexor tendon. The same amounts of protein in the extract from each region were applied and immunostained with anti-human dermatan sulfate proteoglycan antibody before (A) and after (B) chondroitinase ABC treatment: lane 1, myotendinous region; lane 2, middle metacarpal region; lane 3, osteotendinous region.

Fig. 3

Electron micrographs showing cross-sections of collagen fibrils in the myotendinous junction (MTJ), middle metacarpal (MM) region, and osteotendinous junction (OTJ) in the superficial digital flexor tendon. Center-to-center and surface-to-surface distances between collagen fibrils were measured; means are shown in Table 1. Bar: 200 nm.

Fig. 4

Electron micrographs showing longitudinal sections of collagen fibrils and dermatan sulfate (DS) filaments in the myotendinous junction (MTJ), middle metacarpal (MM) region, and osteotendinous junction (OTJ) in the superficial digital flexor tendon. (A) The samples were stained with cupromeronic blue to examine DS filaments. The lengths of the stained DS filaments were measured and the means are shown in Table 1. Arrows indicate DS filaments. Bar: 100 nm. (B) The samples were stained with cupromeronic blue, and then with uranyl acetate. The association angles between the longitudinal axes of the DS filaments and the collagen fibrils were measured and the means for each region are shown in Table 1. The angle was expressed as a value from 0 to 90 °C. Arrowheads indicate DS filaments. Bar: 100 nm.

Fig. 5

A model showing the graded organization of collagen fibrils in the superficial digital flexor tendon. In the myotendinous junction (MTJ) region (upper part of the tendon), collagen fibrils with a small diameter make firm contact with and fuse directly to the basement membrane of muscle fibers. Fine fibrils in the MTJ extend downwards and fuse with each other to form thick fibrils in the middle metacarpal (MM) region (middle part). In the osteotendinous junction (OTJ) region, thick fibrils are predominant and fuse directly to the bone (lower part).