Number of fiber bundles in the fetal anterior talofibular ligament

For the anterior talofibular ligament (ATFL), a three-fiber bundle has recently been suggested to be weaker than a single or double fiber bundle in terms of ankle plantarflexion and inversion braking function. However, the studies leading to those results all used elderly specimens. Whether the difference in fiber bundles is a congenital or an acquired morphology is important when considering methods to prevent ATFL damage. The purpose of this study was to classify the number of fiber bundles in the ATFL of fetuses. This study was conducted using 30 legs from 15 Japanese fetuses (mean weight, 1764.6 ± 616.9 g; mean crown-rump length, 283.5 ± 38.7 mm; 8 males, 7 females. The ATFL was then classified by the number of fiber bundles: Type I, one fiber bundle; Type II, two fiber bundles; and Type III, three fiber bundles. Ligament type was Type I in 5 legs (16.7%), Type II in 21 legs (70%), and Type III in 4 legs (13.3%). The present results suggest that the three fiber bundles of the structure of the ATFL may be an innate structure.


Introduction
Injury to the lateral ligaments of the ankle is one of the most frequent sports injuries in both competitive and recreational sports. Roughly 70% involve injuries to the anterior talofibular ligament (ATFL) alone [8,19,24]. The morphological features of the lateral ligaments of the ankle are thought to be heavily involved in these injuries, and many anatomical studies of the ATFL in particular have been reported [11].
The main morphological features of the ATFL that have been investigated are the number of fiber bundles, fiber bundle length, fiber bundle width, and fiber bundle angle. However, differences in the number of fiber bundles have yet to be clarified. ATFL types with one, two, or three fiber bundles have been reported [12,21], but other reports have suggested that types with three bundles do not exist [2-4, 13, 16, 20, 23, 25]. On the contrary, many recent reports using a large number of specimens have described three fiber bundles [5,7,9,10].
The ATFL is reported to serve a major function in ankle plantarflexion and inversion control [15]. During plantarflexion, the superior fiber bundle tenses and the inferior fiber bundle relaxes, while during dorsiflexion, the superior fiber bundle relaxes and the inferior fiber bundle tenses [22]. In earlier studies, the ATFL was mainly described as the two fiber bundle type, and functions of the superior and inferior fiber bundles differed. In other earlier studies, however, fiber bundle length and total fiber bundle width were the same even when the number of fiber bundles differed, so functional differences were considered unlikely [22]. In all of these reports, the effects of differences in ATFL number of fiber bundles on function remained speculative. In recent studies, three-dimensional reconstructions of single, double, and triple fiber bundles of ATFL have been created. These were used to simulate and calculate ATFL strain during dorsiflexion (20°) and plantarflexion (30°) on the talocrural joint axis and inversion (20°) on the subtalar joint axis. The results suggested that an ATFL with three fiber bundles was weaker than those with single or double fiber bundles in terms of ankle plantarflexion and inversion braking function [6]. However, all specimens used in those previous studies were from elderly individuals. Whether the difference in fiber bundles represents a congenital or an acquired morphology is important when considering the prevention of ATFL damage. The purpose of this study was, therefore, to classify the number of fiber bundles of the ATFL in fetuses.

Cadavers
The ligaments investigated were obtained from 30 legs of 15 Japanese fetuses (mean weight, 1764.6 ± 616.9 g; mean crown-rump length, 283.5 ± 38.7 mm; 8 males, 7 females). All specimens were fixed and preserved in 10% formalin and placed in water for 24 h before dissection. The human fetuses used in the present study belong to the Kyoto Collection of Human Embryos [14,18] and all were Japanese. They were obtained through collaborating obstetricians and in accord with the Japanese Eugenic Protection Law (the present Law for the Protection of Mothers' Bodies), mainly from healthy parents, rather than due to any clinical reasons of either embryos/fetuses or parents. Therefore, these embryos/fetuses can be considered to be representative of the normal Japanese intrauterine population. There were no congenital disorders in the central nervous system in the present cases. Mean gestational age of specimens was 23 weeks (range 21-25 weeks; all in the second trimester) as calculated from the crown-rump length [17]. This study was approved by the ethics committee at our institution (approval no. 4881).

Methods
In the ATFL dissection procedure, the skin, subcutaneous tissue, and crural fascia were removed, and the ATFL was carefully dissected (Fig. 1). The ATFL was then classified by the number of fiber bundles: Type I, one fiber bundle; Type II, two fiber bundles; and Type III, three fiber bundles.

Discussion
This study examined differences in the number of ATFL fiber bundles in the fetus. To the best of our knowledge, no previous anatomical studies have reported on the number of ATFL fiber bundles in the fetus.
The ATFL is reported to serve a major function in ankle plantarflexion and inversion control [15]. One previous study [6] created three-dimensional reconstructions of the single, double, and triple fiber bundles of ATFL. The results suggested that a structure with three fiber bundles yielded an ATFL that was weaker than single or double fiber bundles in terms of ankle plantarflexion and inversion braking function. Our findings indicate that the three fiber bundles of the structure of ATFL may represent innate structural characteristics of the ligament. We, therefore, suggest that the presence of three fiber bundles in the ATFL may represent a risk factor for ATFL injury. In the future, we believe it will be necessary to perform biomechanical research using our basic data with in vivo samples, which may lead to lead to development of the prevention of the ATFL injuries.
Some limitations to this study need to be considered. First, this study investigated only fetal ATFL in the second trimester. Investigations of fetal ATFL in the first trimester have also been attempted, but dissections proved difficult. Second, this study focused solely on differences in the number of ATFL fiber bundles and did not include quantitative analyses. Third, this study did not investigate bone morphology information such as the radius of curvature of the talus [1].

Conclusion
This study examined differences in the number of ATFL fiber bundles in fetuses. ATFL fiber bundles were classified as Type I in 5 legs (16.7%) Type II in 21 legs (70%), and Type III in 4 legs (13.3%). These results suggest that the three fiber bundles of the structure of the ATFL may be an innate structure.