Anatomical-histological study of the Achilles tendon: the search for subtendons

The human Achilles tendon (AT) has recently been described as comprising three morphologically distinct subtendons (STs) originating from respective triceps surae muscles. However, their existence remains controversial. This study aimed to histologically investigate the presence of STs in human AT. We hypothesized that STs are separated by greater distances than fascicles.

Proximal and distal samples collected from 27 human ATs yielded 47 sections, which were processed with Masson's trichrome staining. Forty distances separating tendon subunits were measured per sample using NDPview2. Frequency analysis of these was used to assess hierarchical organization. Three examiners independently visually assessed the samples for the presence of STs. Interobserver agreement and concordance with distance analysis were evaluated using Fleiss' kappa.

Frequency distribution analysis revealed that seven samples (15%) exhibited three STs, and 14 samples (30%) displayed two STs. Overall, 1,850 distances showed a unimodal distribution with a single peak at 0-50µm, corresponding to inter-fascicular distances. Visual assessment identified two STs in 9 to 14 sections (19-30%) and three STs in 3 to 15 Section (6-32%), depending on the examiner. Interobserver agreement was low, with Fleiss' kappa of 0.07 for two STs and 0.096 for three STs. Across all four assessments, kappa was 0.67 for two STs and 0.009 for three STs.

Standard histological techniques applied in our study could not identify STs in histological sections of ATs from elderly subjects. We suggest performing a similar analysis on non-frozen ATs from younger subjects to confirm these findings.