Ankle sprains are among the most common sports injuries. Among the ligaments of the ankle joint, the anterior talofibular ligament (ATFL) is the most prone to injury [1-7]. Ankle sprains frequently recur, which can lead to chronic ankle instability (CAI) that adversely affects daily life and sports activities. Operative stabilization of the ankle joint has been recommended for patients with CAI to reduce pain and restore function [3, 5, 8-14]. Moreover, ultrasound imaging has been reported as a useful modality for the noninvasive evaluation of torn ligaments in reference to the bony landmarks and the fibrillar pattern of the ligament [15-30]. However, the best imaging method for ATFL evaluation has not been clearly defined, which is of concern because without standardization, the results of evaluations are not repeatable. To ensure anatomically accurate surgery and ultrasound imaging of the ATFL, anatomical knowledge of bony landmarks for the ATFL attachment to the distal fibula is required.

Previously, the anterior tubercle (AT) and the inferior tip of the fibula (IT) have been used as bony landmarks to identify ATFL attachments [1, 3, 5, 9, 31-35]. In addition, the fibular obscure tubercle (FOT) has been described as the bony morphology at the anterodistal part of the fibula that divides the attachment of the ATFL and calcaneofibular ligament (CFL) [36, 37]. In ultrasound imaging, the ATFL can be visualized as a distinct fibrillar pattern and relationship with bony landmarks, such as the anterodistal aspect of the fibula and FOT. However, to establish a more precise, repeatable, and easy-to-use method of ATFL imaging, a more obvious landmark is required, which should indicate the ATFL attachment itself.

According to Wolff’s law [38], mechanical forces continue to influence bone construction in the adult body. In addition, some reports have suggested that tensile stresses from dense connective tissues such as ligaments, muscular aponeuroses, and tendons affect bone morphology [39, 40]. We therefore hypothesized the presence of a bony morphology on the anterodistal part of the fibula corresponding to the ATFL attachment site. The primary purpose of the present study was to anatomically investigate the ATFL attachment to the fibula using bone morphology, macroscopic, and histological methods. The secondary objective was to visualize the ATFL attachment to the fibula using ultrasonographic imaging in living specimens based on the anatomical findings obtained in the present study.

Cadaveric specimen preparation and micro-computed tomography imaging

Ethical approval was obtained from the institutional review board of the Tokyo Medical and Dental University (M2020-382). In the present study, 36 feet (9 pairs, 18 halves; 18 left and 18 right feet) from 27 cadavers (average age at death 85.52 [65–104] years, 12 men and 15 women) were used. All cadavers were donations to the Department of Anatomy of the Tokyo Medical and Dental University.

All specimens were fixed in an 8% formalin solution, soaked in 30% ethanol, and stored. Ankles were obtained by cutting 5 cm proximal to the lateral malleolus and distal to the Lisfranc joint using a diamond band pathology saw (EXACT 312; EXAKT Advanced Technologies). The ankles obtained were sagittally trimmed at the center of the tibia, and the medial sides were discarded. The skin and subcutaneous soft tissue were thoroughly removed. Next, we captured images of all lateral ankle halves using micro-computed tomography (micro-CT) (inspeXio SMX-100CT; SHIMADZU) with 200-µm resolution. We reconstructed three-dimensional (3D) images using image analysis software (VG studio MAX 2.0; Volume Graphics GmbH Heidelberg) (Fig. 1A–C). We excluded 9 feet with obvious bone deformities.

Figure 1. The bony morphology of the fibula. Bone morphology of the distal fibula of the right ankle as shown by (A–C) micro computed tomography and (D–F) chemically debrided bone. (A) Micro-computed tomography image. (B) Region boxed in white in (A); dotted line: ridge between attachments of the anterior talofibular ligament (ATFL, dagger) and the calcaneofibular ligament (CFL, asterisk). (C) The border indicated in B, which is remarkable in the anterolateral view of the distal fibula. (D–F) The same specimen as in (A–C) after chemical debridement of soft tissues. ATFL, anterior talofibular ligament; CFL, calcaneofibular ligament; Prox, proximal; Ant, anterior; FOT, fibular obscure tubercle.

To confirm the correspondence between the 3D-CT image and the actual bony surface without dissection artifacts, soft tissues were removed using an 0.9% sodium hydroxide solution (Wako Pure Chemical Industries) in three specimens (Fig. 1D–F). In the remaining 24 specimens, 17 and 7 ankles were randomly assigned for macroscopic and histological analyses, respectively.

Macroscopic observation

Seventeen ankles were used for the gross anatomical analysis. The superior extensor retinaculum, peroneus tertius, extensor digitorum longus, extensor digitorum brevis, and the long and short peroneal muscles were removed. The synovial capsule was removed and the anterior inferior tibiofibular ligament, lateral talocalcaneal ligament, CFL, and ATFL were identified. The tibia and fibers covering the CFL at the distal part of the ATFL were removed to expose the CFL attachment area (Fig. 2A). The lateral talocalcaneal ligament and connecting fibers were removed to confirm the fibrous orientations of the ATFL and CFL (Fig. 2B). The CFL fiber was detached from the calcaneal and fibular attachments (Fig. 3A). Finally, the ATFL was detached from the talar and fibular attachments to reveal its fibular attachment (Fig. 3B). The width, length, and footprint of the ATFL attachment were measured, as well as the distance from the AT and the IT to the margins of the ATFL attachment (Fig. 4).

Figure 2. Fibrous orientations of the ATFL and CFL in their fibular attachments. (A) After removal of the skin, subcutaneous tissues, muscles, synovial articular capsule, and tibia, the anterior talofibular and calcaneofibular ligaments (ATFL and CFL, respectively) in the lateral ankle can be seen. (B) After the removal of connecting fibers, the fibrous orientations of the ATFL and CFL are clearly seen. ATFL, anterior talofibular ligament; CFL, calcaneofibular ligament; Prox, proximal; Ant, anterior.

Figure 3. Fibular attachments of the ATFL and CFL. Shown is the lateral aspect of the right ankle seen in . (A) After removal of the calcaneofibular ligament (CFL) fibers, demonstrating the CFL attachment (asterisk). (B) After removal of the anterior talofibular ligament (ATFL) seen in A, demonstrating the ATFL attachment (dagger) and the posterior talofibular ligament (PTFL) distal to the ATFL and CFL attachments. ATFL, anterior talofibular ligament; CFL, calcaneofibular ligament; Prox, proximal; Ant, anterior.

Figure 4. Measurements of the ATFL attachment. Explanation of the data provided in Table 1. Dotted areas indicate the attachments of the anterior talofibular ligament (ATFL, dagger) and calcaneofibular ligament (CFL, asterisk). (a) The proximal-distal length, (b) the anteroposterior width, (c) the distance between the anterior tubercle of the fibula (section) and the proximal edge of the ATFL attachment, and (d) the distance between the ATFL attachment and the inferior tip of the fibula (double dagger). ATFL, anterior talofibular ligament; CFL, calcaneofibular ligament; Post, posterior; Dist, distal.

Histological analysis

A diamond band saw (EXACT 312) was used to cut parallel to the fiber run and through the AT of the distal fibula, from which slices were made at approximately 5-mm intervals to the distal end of the fibula. After post-fixation with 10% formalin, the slices were decalcified in Plank-Rychlo solution (AlCl₃〮6H₂O [126.7 g/L], HCL [85.0 ml/L], and HCOOH [50.0 ml/L]; FUJIFILM Wako Pure Chemical Corporation) for 1 week, and immersed in 70%, 80%, 90%, and 100% ethanol solution and xylene for 1 day each. The specimens were then placed in a paraffin solution in a mold under vacuum conditions for 3 days and paraffin blocks were created. Serial sections of histological sections were made every 500 µm with a thickness of 5 µm. Masson’s trichrome staining was performed to analyze the bone morphology and attaching fibers of the ATFL (Fig. 5).

Figure 5. Histological analyses of the ATFL attachment by Masson trichrome stain. Sections are parallel to the fibrous orientation of the ATFL. (A) White lines show the orientations and locations of sections. (B) Shown is the proximal part of the ATFL. (C) Magnified view of the region boxed in black in (B); black arrow: the bony ridge at the posterior edge of the ATFL. (D) The distal part of the ATFL. (E) Magnified view of the region boxed in black in (D). The white arrow indicates the bony ridge dividing the attachments of the ATFL and calcaneofibular ligaments (CFL, black arrowheads). ATFL, anterior talofibular ligament; CFL, calcaneofibular ligament; Prox, proximal; Ant, anterior; Lat, lateral.

Ultrasonographic imaging in living specimens

Ten feet of five healthy living specimens (average age 25.6 [23–35] years, 5 women) were investigated. We recruited individuals with no history of previous ankle surgery. The study design obtained ethical approval from the institutional review board of the Tokyo Medical and Dental University (M2022-229). The study requirements, benefits, and risks were explained to all potential participants, and those who desired to participate gave their written informed consent. The aplio i900 scanner (Canon medical systems) with a 5–18 MHz linear transducer was used for ultrasonographic imaging. The participants lay supine on the bed with the knee joint of the test leg flexed to 90 degrees putting their soles on the bed, and 10 to 15 degrees internal rotation.

First, the transducer was placed parallel to the short axis of the fibula to identify the AT (Fig. 6B). Second, the transducer was moved distally to confirm the bony ridge as the posterior margin of the ATFL attachment (Fig. 6C). Third, the transducer was adjusted to visualize the fibrillar pattern of the ATFL using the bone ridge as the reference (Fig. 6D).

Figure 6. Ultrasonographic imaging of ATFL in living specimens. (A) Gray boxes show the locations of ultrasonographic images (B, C), and (D). Dotted areas indicate the attachments of the anterior talofibular ligament (ATFL, dagger) and calcaneofibular ligament (CFL, asterisk). (B) The short axis view at the level of the anterior tubercle (AT, section mark). The anterior tibiofibular ligament (AiTFL) was identified anterior to the AT. (C) The short axis view at the level of the posterior margin (dot) of the attachment of the anterior talofibular ligament (ATFL, white arrowheads) (D). The view at the level parallel to the ATFL fibers. ATFL, anterior talofibular ligament; Post, posterior; Dist, distal.

Statistical analysis

Two raters (Hitomi Fujishiro and Mizuki Azumaya) independently measured the ATFL dimensions, and an intraclass correlation coefficient was computed to assess the accuracy of the measurements. Statistical analyses were conducted using the R software (version 4.2.2; R Foundation for Statistical Computing).

Bony morphology of fibula

Based on the micro-CT imaging of all specimens, the ATFL and CFL attachments were revealed as adjacent impressions at the antero-distal end of the fibula (Fig. 1A–C). The two attachments were separated by a bony ridge that continued distally to the FOT. These impressions and ridges between the ATFL and CFL attachments were confirmed with chemically debrided actual bones (Fig. 1D–F).

Fibrous orientations of the ATFL and CFL and their attachments to the fibula

At the fibular attachments, the directions of the ATFL and CFL fibers were anterodistal and posterodistal, respectively. The directional difference between the ATFL and CFL fibers clearly demarcated the border between the ATFL and CFL attachments (Fig. 2). The inferior fibers of the ATFL could be differentiated from the superior fibers because the former were superficially attached to the posterior talofibular ligament (PTFL) (Fig. 3). The distal margins of the ATFL and CFL attachments were formed by the attachment of the PTFL (Fig. 3B).

The proximal-distal length and anteroposterior width of the ATFL attachment were 11.2±1.9 mm and 4.6±1.6 mm, respectively (Fig. 4, Table 1). Along the anterior edge of the distal fibula, the proximal edge of the ATFL attachment was 11.3±3.1 mm distal to the AT and the distal edge of the ATFL attachment was 11.0±1.4 mm proximal to the IT.

Table 1 . Measurements on the ATFL attachment (n=17)

Measurements location	Mean (mm)	SD	ICC	95% CI
Length of the ATFL attachment (a)	11.2	1.9	0.983	0.961–0.993
Width of the ATFL attachment (b)	4.6	1.6	0.989	0.975–0.996
Length from the AT to proximal edge of ATFL (c)	11.3	3.1	0.990	0.977–0.996
Length from the IT to distal edge of ATFL (d)	11.0	1.4	0.959	0.909–0.984

The measurements are demonstrated in Fig. 4. ATFL, anterior talofibular ligament; AT, anterior tubercle of the fibula; IT, inferior tip of the fibula; ICC, intraclass correlation coefficient; CI, confidence interval.

Histological analysis

At the proximal part of the ATFL attachment, the superior fibers of the ATFL were attached anteriorly to the bony ridge via the fibrocartilage structure (Fig. 5A–C). At the distal part of the ATFL attachment, the inferior fibers of the ATFL were attached anteriorly to the CFL attachment less compactly than the superior fibers (Fig. 5D, E).

Ultrasonographic imaging of the ATFL in living specimens

At the level of the AT, the short axis view visualized the attachment of the anterior tibiofibular ligament as the hypoechoic part anterior to the AT (Fig. 6B). At the level of the posterior margin of the ATFL attachment, the short axis view visualized the ATFL fibers attached anteriorly to the bony ridge as the hypoechoic part (Fig. 6C). By adjusting the plane of transducer, the ATFL fibers were visualized as the hyperechoic fibrillar pattern anterior to the bony ridge (Fig. 6D).

This study revealed that a bony ridge is present at the boundary between the ATFL and CFL fibular attachments. These attachments could be distinguished by the difference in fiber orientation. Histologically, the ATFL was attached to the anterodistal part of the fibula via the fibrocartilage anterior to the bony ridge, which indicated the border with the CFL attachment.

To clarify and identify the ATFL attachment, its distance from the AT and IT has been described in previous papers [1, 3, 5, 9, 31-35]. Taser et al. [1] and Haymanek et al. [32] reported that the distances from the AT to the center of the ATFL attachment were 20 and 17 mm, respectively. The distance from the IT to the center of the ATFL attachment was reported as approximately 10–16 mm [1, 3, 31, 34, 35]. Recently, Buzzi et al. [36] identified a tubercle as the bony border between the ATFL and CFL attachments. Matsui et al. [37] recently termed it the FOT and suggested its use as a landmark for the surgery of CAI patients. The distance from the FOT to the center of the ATFL has been reported as 3.7 mm [13, 37, 41]. However, these three markers, as described above, were not bony morphologies that directly represented the ATFL attachment, but merely adjacent bony structures. In the present study, the bony morphology of the ATFL attachment was confirmed as an impression at the anterodistal part of the fibula, and a bony ridge running from the FOT to the proximal region was identified as the border between the ATFL and CFL attachments. The bony ridge of the ATFL attachment could be a new, direct landmark for surgery and ultrasonographic imaging.

Previous studies have disagreed regarding the fibrous connection between the ATFL and the CFL at the fibular attachments. Some papers reported that the ATFL and CFL fibers were distinct [42, 43]. In contrast, others reported the existence of connective fibers running between the ATFL and CFL, which covered the inferior part of the ATFL and the anterior part of the CFL [4, 14, 44, 45]. In the present study, we demonstrated that the fibrous orientations of the ATFL and CFL were distinct, and that the ATFL and CFL attachments were clearly distinguished by an intervening bony ridge. Based on the results of the present study, the discrepancy regarding the fibrous connection between the ATFL and CFL could be explained by differences in the observed layers and locations, that is, the deep layer, composed of dense connective tissues, versus the superficial layer, composed of loose connective tissues.

The results of this study have several clinical implications. Our results could improve the reliability of ultrasonographic visualization of the ATFL. In previous studies, the anterolateral aspect of the fibula [16, 18, 21, 23, 25, 26, 30] and FOT [5, 27] have been used for ATFL identification during ultrasonography. However, as a bony landmark for ultrasonographic imaging, the anterolateral aspect of the fibula is unspecific and outside the actual ATFL attachment. The FOT corresponds with the attachment of only the inferior fibers of the ATFL, because it is located at the distal end of the ATFL attachment. In particular, the inferior fibers have a looser composition than the superior fibers and are difficult to evaluate correctly in ultrasonographic imaging. In the present study, we discovered a bony ridge that corresponds to the posterior margin of the ATFL attachment itself. This ridge was obvious, and the superior fibers of the ATFL were directly attached anteriorly to the ridge. We also visualized the ATFL as a hyperechoic fibrillar pattern by ultrasonographic imaging based on the bony ridge on the anterodistal edge of the fibula. This bony ridge could be a valuable, easy-to-use landmark for ultrasound imaging of the ATFL attachment if combined with the identification of the fibrillar pattern of the ATFL. According to the biomechanical study, lateral ankle laxity due to ATFL deficiency could be reduced by combined ultrasound-guided ATFL repair and augmentation [46]. This bony ridge may also support the identification of the anatomical attachment of the ATFL on the fibula during the ultrasound-guided repair.

This study had several limitations. First, the observation using ultrasonographic imaging was performed only on healthy individuals and only women. Second, the sample size of ultrasonographic imaging was small. Therefore, in vivo ultrasonographic imaging of more living volunteers and clinical cases with ankle sprains will be necessary to validate our findings.

In conclusion, the ATFL and CFL attachments were observed as adjacent impressions at the anterodistal end of the fibula, which were separated by a bony ridge. This bony ridge may be a new landmark for ultrasonography.

We acknowledge and thank the anonymous individuals who generously donated their bodies for this study.

Conceptualization: AN, KA, SH. Data acquisition: HF, MA. Data analysis or interpretation: HF, AN, MA. Drafting of the manuscript: HF, AN. Critical revision of the manuscript: OH, KA, SH. Approval of the final version of the manuscript: all authors.

No potential conflict of interest relevant to this article was reported.