The process by which individual triple helical collagen molecules assemble into mesoscopic structures (1-51-micron length fibrils with a regular axial periodic “D-banding” pattern that is independent of fibril diameter-remains an intriguing conundrum. The current, “accepted” model of tendon collagen (6) considers the characteristic “67 nm repeat” as being formed from a quarter staggered, side-by-side alignment of five triple helices(7,8), which was initially proposed by the early work of Hodges and Petruska (9). Essentially two-dimensional, this interpretation has several deficiencies that earlier theoretical work tried to address (10-12). One model suggested a layered, spiral arrangement of collagen molecules (13), though experimental data to support such a contention was lacking. The existing models for the supramolecular structure of collagen fibrils presented in a review by Jäger and Frutzl (14), fail to explain how D-banding is preserved independent of fibril diameter; how collagen fibrils “grow” with pointed ends (15,16); why the surface of collagen fibrils is not flat but corrugated, with indentations at the D-band; and how fibrils (consisting of a few molecules and up to 10 nm in diameter) form into fibrils of greater diameter (from 50 to several hundred nm) (16,17) and thence into macroscale objects (e.g., tendons). Collagen structure thus follows a well-established principle in biology-that tissue form reflects functional requirements (18). Thus, Wolff’s Law for bone expostulates that mechanical usage drives skeletal structure; likewise, variable ratios of fast and slow myofibrils are found in skeletal muscle undergoing differing amounts of work. Similarly, the spiral or twisted rope features observed by atomic force microscopy (AFM) are the nanoscale equivalent of microscopic crimps formed by relaxation of the subcomponents (plies) that make up collagen fibrils of tendon and contribute to some of their mechanical features (19). Although early transmission electron microscopy and freeze-fracture studies demonstrated the possible spiralization of the collagen structure, as presented in the pioneering work of Ruggeri (18,19), the interdependence between this behavior of the fibril and the consistent periodicity along its length remained unclear.

In this study, we provide AFM data that supports this twisted structure-in particular, we stress the attributes of the fibril morphology that make its structure intriguingly similar to a classical “rope”. We then take these observations at face value by applying a mechanical rope model, taking advantage of recent progress in the mechanical modeling of rope structure in such areas as textile yams and DNA supercoiling (20-23). Although previous studies discussed the possible generation of a rcpeatable periodicity along a fibril, none considered the variation of the fibril diameter (20,21). We show that the rope hypothesis is consistent with experimental observations by taking reasonable values for the model parameters. In particular, the produced D-banding is found to be independent of the fibril diameter. In addition we observe that the model is not sensitive to the choice of parameters. The predicted rope angle is continued experimentally and provides an independent test of the model.

MATERIALS AND METHODS

Sample preparation

A suspension of native bovine digital tendon collagen fibrils was (Ethicon, Somerville, NJ) was dialyzed at 1 mg/ml against 10 mM acetic acid before use and stored at 4°C. This preparation has been used extensively in platelet function studies, for example (22). For topological assessment by atomic force microscopy, a sample was prepared by deposition of a 20 µl droplet of the Mock solution (1 mg/ml) onto APTES-treated glass slides. A typical incubation time of

Atomic force microscopy

Commercial atomic force microscopes (Dimension 3000, Veeco, Santa Barbara, CA; JPK Nanowizard, Berlin, Germany) were used in contact mode (NPS tips, Veeco) to record both topologic (height) and error signal (deflection) images.

EXPERIMENTAL RESULTS

Topological diversity of collagen fibrils

We studied the topology of tendon collagen fibrils directly by AFM as shown in Fig. 1. Low-resolution images (Fig. 1, b and c) provide an overview at the micron scale of the nalure and diversity of the fibrils obtained from digital tendon when compared with fibrils from tail tendon (Fig. 1 d). Fibrils from tail tendon, a relatively mechanically unloaded tissue, are more uniform in structure and generally straight over the length scale examined (23). In contrast, fibrils from the load-bearing digital tendon are heterogeneous and can be classified into two populations depending on whether or not a repeatable irregularity (spiral or twisted features) is observed along the length of the fibril (observed in 32% of the fibrils studied, n = 296). These are the nanoscale homologs of “crimps” mat are characterized as wavy structures in light microscopic histology (24,25). The mechanics at low-strain range (