2.1

Twenty embalmed cadaveric feet, each preserved in continuity with the whole body, were obtained from 11 specimens (mean age: 76.9 ± 5.1 years, 5 female and 6 male) donated to the Jacques Lisfranc Faculty of Medicine.

Specimens with any evidence of previous surgery, trauma, osteoarthritis, rheumatologic conditions, ligament injuries, or range of motion limitations were excluded following macroscopic examination. The study was approved by the Institutional Review Board (IRBN762024/CHUSTE) and conducted in accordance with the ethical standards of the Declaration of Helsinki.

2.2

The bodies were embalmed using the Graz method and stored at 4°C. Two hours prior to testing, specimens were brought to room temperature . No prior manipulation that could affect joint integrity was performed. The lower limbs were left intact to preserve the proximal muscular and tendinous insertions, thereby maintaining the periarticular environment of the ankle and subtalar joints.

2.3

The testing protocol followed a previously described method . Four custom-made supports were rigidly fixed to the tibia, fibula, talus, and calcaneus using 3 mm bicortical pins, each bearing three retroreflective markers. Three bony landmarks were palpated on each bone to create triangular solid models based on the Rizzoli model and ISB guidelines ^, .

For the tibia and fibula, landmarks included the tibial spine center, medial malleolus, and lateral malleolus ^, . On the talus, landmarks were the uppermost points of the medial and lateral talar dome and the intersection of the talar neck axis with the articular surface of the talar head, identified via anterior arthrotomy. For the calcaneus, the selected points were the uppermost aspect of the sustentaculum tali, the most prominent point of the peroneal tubercle, and the superior edge of the posterior tuberosity, accessed through a small targeted incision ( Fig. 1 ) .

Illustration of bony landmarks used for bone talus and Calcaneus modelling, Calcaneus was modelled as a triangular solid (red lines) based on the three bony landmarks (red dots): sustentaculum Tali, peroneal tubercle, upper edge of great tuberosity illustrated on an upper and lateral view. Talus was modelled as a triangular solid (yellow lines) based on the three bony landmarks (yellow dots): intersection between talar neck axis (blue dashed arrow) and articular surface, medial aspect of talar dome, lateral aspect of talar dome illustrated on an upper view of the talus.

CFL and ATFL footprints were exposed through careful dissection by an experienced orthopedic surgeon to minimize soft tissue disruption ( Fig. 2 ) . Segment motion of the tibia, fibula, talus, and calcaneus was recorded, and changes in CFL and ATFL insertion distances were analyzed to assess ligament strain under different SJ ligament conditions.

Illustration of footprint pointed used for assessing distance between proximal and distal insertion of ATFL and CFL. The proximal footprint of ATFL and CFL are illustrated by respectively a green and yellow dot on a lateral view of a fibula. The distal footprint of ATFL is represented by a green star on a lateral view of a talus and the distal footprint of the CFL is represented by a yellow star on a lateral view of the calcaneus.

Positional data were recorded at 100 Hz using eight synchronized high-definition Kestrel 2200 cameras (Motion Analysis Corp., Santa Rosa, CA, USA) surrounding the calibrated volume. Static and dynamic calibration was performed before each specimen test. Segment motions were reconstructed using Cortex 9.5 software (Motion Analysis Corp.), with post-calibration residual error maintained below 0.15 mm.

2.4

Three cycles of varus/valgus loading and flexion/extension were performed before testing to release soft tissue adhesions. Three tests were conducted on each foot with the tibia held fixed, performed by a single orthopedic foot and ankle surgeon. First, a manual anterior drawer test (ADT) combining anterior translation and internal rotation assessed translational and rotational motion of the ankle and SJ. Second, a varus tilt test in neutral ankle position (VTTN) evaluated coronal-plane motion of both joints. Third, a varus tilt test in maximal dorsiflexion (VTTF) was used to neutralize talar motion within the mortise and isolate SJ movement ^, . ( Fig. 3 )

Illustration of a varus tilt test performed on an ankle in neutral flexion.

Each test was repeated under four ligament conditions, simulating progressive injury severity according to the typical injury pattern following a LAS . Testing began with non-injured ankles. At the second stage, the ATFL was sectioned. At the third stage, specimens were randomized into two groups: the extrinsic group (CFL sectioned) and the intrinsic group (CL + ITCL sectioned). At the fourth stage, the remaining ligaments were sectioned (CFL in the intrinsic group; CL + ITCL in the extrinsic group). Each test was repeated three times under each ligament condition ( Fig. 4 ) .

Illustration of the experimental protocol. For each ligament condition, each test was repeated three times. Ligament sectioning followed a progressive severity model. At the third stage, specimens were randomly assigned to one of two groups based on the ligament cut at that step. If the CFL was cut, the specimen was assigned to the intrinsic group; if the CL and ITCL were cut, the specimen was assigned to the extrinsic group. ATFL-: Anterior tibio-fibular ligament section; CFL-: Calcaneo-fibular ligament section; CL-: Cervical ligament section; ITCL: interosseus talo-calcaneal ligament section; ANT: combined anterior and internal rotation test; VTTN: varus test on ankle in neutral flexion; VTTF: varus test on maximal dorsiflexion .

Reconstructed opto-electronic marker data were filtered using a 4th-order Butterworth filter (6 Hz cutoff). Palpated landmarks were used to reconstruct bony segments. The 3D rotation sequence was: dorsiflexion/plantarflexion (F/E, positive for dorsiflexion), internal/external rotation (IR/ER, positive for internal), and varus/valgus (Var/Val, positive for varus). Talus motion was expressed relative to the tibia (tal/tib), and calcaneus motion relative to both tibia (cal/tib) and talus (cal/tal). Distances between ATFL and CFL insertion points were also recorded.

The neutral position was defined as the starting position of the first ADT on the intact ankle, with zero rotation in all planes. Each test was repeated three times, and all repetitions analyzed. The most relevant rotation plane was used as the reference (F/E for ADT; Var/Val for VTTN and VTTF). The time points corresponding to the extrema (minimum and maximum) in this reference curve were extracted. These time points were used to determine the minimal and maximal values of the rotational angles, as well as the maximum distances between the proximal and distal insertions of the ATFL and CFL. For each test and ligament status, the difference between maximal and minimal rotation was calculated and used for the results, reflecting the amplitude of rotation from the average neutral position. Maximal distance between the proximal and distal insertions of the ATFL and CFL was calculated and used for analysis reflecting the elongation of the ligament. A greater maximal distance reflects elongation of the ligament, indicating a higher mechanical stress. Finally, the values obtained for the 3 repetitions were averaged to obtain one amplitude value (rotations) and one maximal value (elongation).

2.5

Statistical analyses were performed using SPSS Statistics© (version 28, IBM©, Armonk, NY, USA). Joint motions were assessed using single-factor repeated-measures analysis with within-specimen comparisons. The factor was ligament status: Non-Injured; ATFL−; ATFL/CFL− or ATFL/CL−; ATFL−/CFL−/CL−/ITCL− or ATFL−/CL−/ITCL−/CFL−.

Motion parameters were analyzed according to the test: flexion/extension (F/E) and internal/external rotation (IR/ER) for ADT; varus/valgus (Var/Val) for both VTTN and VTTF. Motion was assessed for three bone pairs: calcaneus–tibia (ankle–SJ complex), talus–tibia (ankle joint), and calcaneus–talus (SJ). Rotation amplitude was used to evaluate joint motion; maximal values were used to assess ligament elongation.

To assess the effect of intrinsic SJ ligament injury on ATFL and CFL strain, changes in insertion distances were analyzed across the four ligament conditions and three test types using the same model.

A significance threshold of p < 0.05 was used for all analyses.

3.1

During the VTTF, varus rotation between calcaneus and talus significantly increased following combined CL and ITCL section in both the extrinsic (3.15° vs 4.36°, +38.4 %, p = 0.025) and intrinsic groups (2.04° vs 3.49°, +71.1 %, p < 0.001). No significant change was observed after CFL section ( Fig. 5 ).

Rotational motions between the calcaneus and talus under different ligament conditions, according to the applied test. The extrinsic group corresponds to a sequential sectioning of the ATFL, CFL, and CL+ ITCL. The intrinsic group corresponds to a sequential sectioning of the ATFL, CL+ITCL, and CFL. Non-Injured: intact ankle with no ligament section; ATFL: anterior talofibular ligament; CFL: calcaneofibular ligament; CL: cervical ligament; ITCL: interosseous talocalcaneal ligament; “−” indicates ligament section. ADT: Anterior Drawer Test; VTTN: Varus tilt test for an ankle in neutral position; VTTF: varus tilt test for an ankle in dorsiflexion; F/E: flexion/Extension; IR/ER: internal rotation/ external rotation; var/val: varus/valgus. The Y-axis is expressed indegrees (°).

3.2

Between the calcaneus and the tibia, FE amplitude increased significantly after ATFL section in both the extrinsic (5.92° vs 8.18°, +38.2 %, p = 0.003) and intrinsic groups (5.03° vs 6.84°, +36.0 %, p < 0.009) during ADT.

During the VTTN, varus rotation increased after ATFL section in the extrinsic group (9.45° vs 10.89°, +15.2 %, p = 0.006), and after CFL section in both extrinsic (10.89° vs 14.25°, +30.9 %, p = 0.002) and intrinsic groups (11.06° vs 15.11°, 36.6 %, p < 0.001).

During the VTTF, varus rotation increased significantly after CL and ITCL sectioning in both groups: extrinsic (3.82° vs 5.70°, +49.2 %, p = 0.006) and intrinsic (2.85° vs 5.20°, +82.5 %, p < 0.001) ( Fig. 6 ) .

Rotational motions between the calcaneus and tibia under different ligament conditions, according to the applied test. The extrinsic group corresponds to a sequential sectioning of the ATFL, CFL, and CL+ ITCL. The intrinsic group corresponds to a sequential sectioning of the ATFL, CL+ITCL, and CFL. Non-Injured: intact ankle with no ligament section; ATFL: anterior talofibular ligament; CFL: calcaneofibular ligament; CL: cervical ligament; ITCL: interosseous talocalcaneal ligament; “−” indicates ligament section. ADT: Anterior Drawer Test; VTTN: Varus tilt test for an ankle in neutral position; VTTF: varus tilt test for an ankle in dorsiflexion; F/E: flexion/Extension; IR/ER: internal rotation/ external rotation; var/val: varus/valgus. The Y-axis is expressed in degrees (°).

3.3

Between the talus and the tibia, FE amplitude increased after ATFL section in both extrinsic (6.62° vs 8.65°, +30.7 %, p = 0.007) and intrinsic groups (6.43° vs 8.20°, +27.5 %, p < 0.009) during the ADT.

During the VTTN, varus rotation increased after ATFL section in extrinsic (3.12° vs 4.22°, +35.3 %, p = 0.002) and intrinsic groups (3.64° vs 4.53°, +24.5 %, p = 0.019) and further increased after CFL section in both extrinsic (4.22° vs 8.24°, +95.3 %, p < 0.001) and intrinsic groups (5.05° vs 8.95°, +77.2 %, p < 0.001) ( Fig. 7 ) .

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