Tendon tissue

The rat tibialis anterior was selected because of its uniform width (∼1.5 mm) throughout its length (∼10 mm). Three mature male Long-Evans rats were killed with carbon dioxide. As significant changes (see below) were already present after examination of the third loaded specimen, we report on three experimental and three control specimens only. The six tibialis anterior tendons were exposed, and the muscle belly of tibialis anterior was separated from surrounding structures so the tendon could be dissected free without direct contact. The tendons were rinsed three times in sterile saline and maintained overnight in Dulbecco’s modified eagle’s medium containing 10% fetal bovine serum and 100 U/ml penicillin, streptomycin, glutamine (DMEM) at 37°C and buffered with 25 mM Hepes buffer. After the overnight incubation, the paratenon could easily be removed by gently pulling with tweezers.

Mechanical loading protocol

The tendon proper was clamped at either end with custom built, toothed aluminium grips and submerged in a heated (37°C) organ bath containing pre-warmed DMEM which was refreshed every 15 minutes. One grip was fixed to a stationary clamp, and the other grip (muscle end) was attached to a moveable arm placed so as to eliminate tendon slack. This yielded a grip to grip length of 6.4 (0.4) mm. The moveable arm was programmed to oscillate in a longitudinal sine wave at 1 Hz, and the strain was gradually increased to 20% of the grip to grip length. This supraphysiological load was maintained for six hours. The right tendon from each of three animals was loaded, and the left served as control and was treated identically except that it was not subjected to any strain.

Light microscopy examination

After the six hour loading protocol, tendons were cut from the grips with a sterile scalpel, rinsed with sterile phosphate buffered saline (PBS) and maintained in DMEM in a 5% CO₂ tissue culture incubator for 24 hours to allow a substantial number of cells to commit to apoptosis. Next, the tissue was fixed in fresh, cold 4% paraformaldehyde for 24 hours, embedded in paraffin and sectioned at 5 μm. Haematoxylin and eosin sections were examined on a Zeiss Axioplan using objectives up to 63× magnification, and digital images were obtained.

Detection of apoptosis

Two immunohistochemical methods were used to examine apoptosis in loaded and control tendon tissue. The first used a monocolonal mouse antibody (F7-26) that labels single stranded DNA breaks (Chemicon, Temecula, California, USA). Tissue sections were deparaffinised in xylene, then washed sequentially in 100%, 95%, and 70% ethanol and PBS. Slides were incubated in PBS containing 0.2 mg/ml saponin and 20 μg/ml Proteinase K at room temperature for 20 minutes to expose the antigen. They were then washed in distilled water and transferred to 50% formamide (v/v distilled water) for 20 minutes to denature the DNA. After blocking with 3% non-fat dry milk, the sections were incubated with F7-26 (10 μg/ml) for 30 minutes at room temperature, then with biotinylated goat anti-mouse IgM (1:1000; Dako) for 30 minutes. Finally, the sections were incubated with avidin-fluorescein isothiocyanate (Sigma, St Louis, Missouri, USA), counterstained with propidium iodide, and mounted with gel mount (Vector, Burlingame, California, USA).

The second method used a rabbit polyclonal antibody that specifically recognises the cleaved form of caspase-3 (Cell Signaling Technology, Beverley, California, USA). The sections were cleared as described above, then permeabilised with methanol for 10 minutes. Endogenous peroxidases were quenched with 3% hydrogen peroxide. The sections were incubated with the primary antibody (1:50) overnight at 4°C, then visualised using an avidin/biotin complex kit with diaminobenzidine as the substrate (Vector) and Harris’ haematoxylin as the counterstain. For both antibodies, one section per slide was incubated with mouse or rabbit serum (rather than the primary antibody) that eliminated the specific antibody staining. To quantify apoptosis from the sections stained with monoclonal mouse antibody (F27-6), one investigator (AS) counted cells from non-overlapping fields in both loaded and control tendon while remaining blinded to the group.

Statistical analysis

All values are expressed as mean (SD). The distribution of cell counts approximated normality. Thus statistical comparison of cell counts in loaded and unloaded tendon was performed using unpaired Student’s t tests. The confidence limit was predetermined at an α level of 0.05.

Description of tendon tissue and macroscopic change

At the end of the six hour protocol, the loaded tendons had lost their normal, tightly packed appearance. The ends immediately adjacent to the grips appeared grossly frayed. They were ignored in the analysis to eliminate the possibility of examining tendon that had been directly injured by the grips.

Light microscopy appearance

The haematoxylin and eosin sections of control tendons showed the typical, tightly packed arrangement of collagen fibres. Most tenocytes were spindle shaped, with sparse cytoplasm, arranged in longitudinal arrays. Superficial regions of the tendon occasionally displayed rounded tenocytes, some of which were located in lacunae. The rat tibialis anterior tendon did not display any vessels or nerves. The haematoxylin and eosin sections of the loaded tendons confirmed that the high strain protocol had induced a stretch overload injury (fig 1A,B⇓). There was evidence of injury to both cells (tenocytes) and collagen fibres. Collagen fibres displayed longitudinal separation as well as transverse tears, with fraying of fascicles. There were three main types of tenocyte abnormalities observed in loaded tendons. The first were regions of complete absence of tenocytes. This was reflected in the lower number of tenocytes observed per viewing field. In the second abnormality, injured tenocytes appeared larger than normal, with prominent cytoplasm and a more polygonal or rounded shape. In the third abnormality, tenocytes appeared shrunken, with fragmented nuclei consistent with apoptosis.

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Figure 1

 Cell death resulting from supraphysiological loading (20% strain for six hours) of the rat tibialis anterior tendon. Haematoxylin and eosin stain after the loading experiment shows the characteristic well organised features of normal tendon (A, control tendon) and the disarray of tendon subjected to loading (B). In the loaded tendon, the blue arrow points to disrupted collagen fibres and the black arrow to nuclear fragmentation, a hallmark of apoptosis. Monoclonal mouse antibody (F27-6) staining for single stranded DNA breaks revealed virtually no apoptotic cells in the control tendon (C) but cells positive for apoptosis in the loaded specimen (D). Finally, a rabbit polyclonal antibody specifically recognising the cleaved end of caspase-3 revealed positive staining localised to the nucleus in loaded tendon (E).

Apoptosis assays

Control tendons showed little or no staining (zero or single positive cells per section) with the F7-26 antibody. In contrast, the loaded tendons displayed numerous apoptotic cells (fig 1C⇑,D). The labelled cells typically colocalised with abnormal nuclear morphology, including nuclear fragmentation or relative loss of nuclear staining. The staining against cleaved caspase-3 showed a similar pattern, with positively labelled cells only detected in the loaded tendons, but the staining localised both to nucleus and cytoplasm (fig 1E⇑). Identical results were also found with tendons that were quick frozen rather than paraffin fixed, and fixed in acetone rather than paraformaldehyde.

Apoptosis quantification revealed that the mean (SD) number of apoptotic cells per field in loaded tendon was 21 (4) whereas in control tendon it was 2 (2) apoptotic cells per field (p  =  0.000). There was no difference in the number of total cells per field in the two conditions (loaded  =  115 (32) compared with unloaded  =  120 (25)). Thus, in the loaded and control tendon, apoptotic cells comprised 20.2% and 1.3% of the cells respectively.