The human zygapophysial joint (ZJ) exhibits regional differences; it has a joint plane between the adjacent articular processes that extends almost horizontally in the cervical region, frontally in the thoracic region, and sagittally in the lumbar region [1, 2]. However, radiologic evidence has shown that the cervical ZJ is situated frontally rather than horizontally in infants and children [3]. Therefore, the lower vertebral element is located antero-inferiorly to the upper element at the ZJ. Although many studies have examined the development and growth of fetal cervical vertebrae [4-7], little is known about the shape, extent, and surrounding tissue in the joint cavity in fetuses and children. Furthermore, observations of the ZJ have not been included in imaging studies of human fetuses [8-10].

In our recent study of the cervical vertebrae [11-13], we found that the morphology of the fetal cervical ZJ is markedly different than that of the adult cervical ZJ. The joint cavity is very large as it extends posteromedially along the vertebral lamina beyond the articular process. Such posterior extension of the ZJ was not observed during our previous studies on the thoracolumbar vertebrae in mid- and late-stage fetuses [11, 14], and it has not been reported in studies on the thoracolumbar vertebrae conducted by other groups [15, 16]. A disruption while observing the ZJ seems to be the sectional plane; the frontal sections are likely to make discrimination between the lamina and articular process difficult. Therefore, this study aimed examine the morphology of the potentially large cervical ZJ and the associated synovial tissue. We also examined the sagittal and horizontal sections of near-term fetuses.

This study was conducted in accordance with the Declaration of Helsinki 1995 (as revised in 2013) and was approved by the Ethics Committee of Complutense University (B08/374). We examined paraffin-embedded histological sections from 20 near-term fetuses (gestational age, approximately 30–38 weeks; crown-rump length [CRL], 250–315 mm). All fetuses were part of the large collection kept at the Department of Anatomy of Complutense University in Madrid, Spain; they were the results of miscarriages and ectopic pregnancies obtained by the Department of Obstetrics of Complutense University. To demonstrate elastic fibers, most sections were stained with hematoxylin and eosin (H&E); however, other sections were stained with elastica-Masson (a variation of Masson-Goldner staining) [17, 18]. All sections were prepared with a 50-µm or 100-µm interval, and sectional planes were sagittal (15 planes) or horizontal (5 planes). Most of the sagittal sections overlapped materials used for our recent studies [11, 12]. The specimens were not available for immunohistochemistry of the smooth muscle actin, which was necessary for the identification of myofibroblasts, due to the lengthy amount of time spent preserved in an acidic formalin solution. An Eclipse 80 microscope (Nikon, Tokyo, Japan) was used to observe and photograph the specimens.

Sagittal sections showed that in the cervical ZJ between the third and seventh vertebrae, the joint cavity of the ZJ extended posteriorly along the vertebral lamina to reach the base of the spinous process (Figs. 1–3). The space between the vertebrae appeared narrow in the anterolateral site (i.e., between the articular processes) (Fig. 1A), but it became wider in the posteromedial sites (i.e., between the laminae) (Fig. 1B). Finally, the space was widest between the bases of the spinous processes because the joint cavity extended along the supero-inferior axis in a wide space between the spinous processes (Fig. 1C). The term “joint” was not suitable for the most posterior part because the cavity does not have bone articulation. Figs. 1A and 3A show the joint cavity between the laminae of the vertebrae and the ZJ between the articular processes of the sixth and seventh vertebrae.

The cervical ZJ consistently had a primitive synovial tissue containing multiple recesses of the joint cavity at the anterior and posterior ends (Figs. 1A, 2A). The deepest or longest recess extended superiorly along the upper adjacent vertebra at the anterior end of the ZJ (Figs. 2C, 3D), whereas the next deepest recess was typically found at the posterior end, where it extended along the lower adjacent vertebra (Figs. 1E, 2E). These deep recesses had a smooth synovial cell lining (Figs. 2C, 3C) and often gave off daughter recesses; the daughter recesses were short and thin. Furthermore, the daughter recess tended to carry rough surfaces because of tiny “capes and gulfs” (Figs. 1D, 2C), and the cuboidal cell lining (Figs. 3C, 4E). In horizontal sections, a deep recess along the upper adjacent vertebra (Fig. 4A, left) indicated the anterior end of the cavity, as seen in sagittal sections. Horizontal sections showed some of the daughter recesses penetrting the thick synovial tissue to reach the lower adjacent vertebral element (Fig. 4B, C). Therefore, the recesses of the joint cavity were likely to create a three-dimensional tree-like configuration in the anterior synovial tissue.

Synovial folds were often seen between the bases of the spinous processes (Fig. 1E) and between the lower cervical vertebrae (Figs. 2E, 3A). A thick periosteum comprising collagen fiber bundles was present at the end of the joint cavity (Fig. 3D, E). No elastic fibers were identified around the joint cavity (Figs. 3C, 4E), with a few exceptions (Fig. 3I); however, the internal elastic lamina of the muscular arteries was well-developed in the same specimen (Figs. 3H, 4H). When compared with the arterial wall, rare elastic fibers around the ZJ were not mature because of the sparse distribution of straight fibers.

Abundant veins or a part of the external vertebral plexus surrounded the ZJ. These veins often had a high, cuboidal endothelium and no or few elastic fibers (Fig. 3F). The venous plexus surrounded the spinal nerve root and ganglion (Figs. 1B, 2A). Notably, at and near the anterior end of the joint cavity, veins created multiple “recesses” or slit-like tributaries to those inserted deeply in the primitive synovial tissue (Fig. 2B, F); they ramified to create a gulf-like appearance similar to that of the joint cavity recess. A cross-section of the venous recesses was often seen at the anterior of the joint cavity (Fig. 2D). It exhibited an impressive morphology similar to that of the arteriovenous anastomosis or a sphincter of the intestine; however, there was no evidence of the presence of smooth muscles and nerves. At the anterior margins, the joint cavity recesses were located close to, or even interdigitated with, specific venous tributaries. We did not perform three-dimensional reconstruction.

The present study demonstrated a specific synovial tissue containing complicated cavity recesses that were interdigitated with or closely located to venous tributaries of the external vertebral plexus (Fig. 5). According to our previous study, it had been specifically found in the subaxial cervical region [13]. The venous-synovial complex was usually seen at the anterior end of the cervical ZJ, and it seemed to be a transient morphology in late-stage fetuses and, possibly, in infants. Based on radiologic evidence, Cattell and Filtzer [3] reported frequent anterior dislocation of the cervical vertebrae in infants and children. The venous-synovial complex might have a critical role in the stabilization of the ZJ in infants until establishment of the ligamentum flavum. Blood in the synovial tributaries of veins likely pushed the joint cavity in the posterior direction. A cross-section of a specific vein in the synovial tissue exhibited an appearance similar to that of arteriovenous anastomosis, which should contain smooth muscles, elastic fibers, arterial branches, and nerves [19-21]. However, we did not find elastic fibers or arterial branches around the synovial vein. The spinal nerve root and ganglion were surrounded by the vertebral venous plexus and, therein, the veins were likely to reduce mechanical stress from the joint movement. Likewise, to avoid injury by the suggested dislocation in infants, a complex of venous tributaries seemed to provide a cushion to the deep recess.

A capsule of the ZJ contained abundant elastic fibers in adults [22]. In the head and neck, until 30 weeks of gestation, elastic fibers appeared in the larynx, pharynx, middle ear, pharyngotympanic tube, and auricle [23], as well as in the ligamentum nuchae behind the cervical vertebrae [11]. To create the ligamentum flavum, elastic fibers should occupy later posterior large parts of the ZJ between the cervical vertebral laminae. Therefore, replacement of the joint cavity by elastic fibers most likely occurs between the cervical laminae in childhood or adolescence. We have previously reported analogies of such a replacement by elastic fibers in aged cricothyroid joints [24] and fetal incudopetrosal joints [25, 26]. The cricothyroid joint is often closed by elastic fibers with aging, whereas the incudopetrosal joint between the incus and petrosal part of the temporal bone changes into the posterior ligament of incus, possibly after birth. These two joints should resist against vibration because collagen fibers are not suitable for the maintenance of joint stability under vibration [27]. Therefore, instead of vibration, a specific “cue” seems necessary for the suggested delayed synthesis of elastic fibers in the cervical ligamentum flavum. Postnatal growth of the uncinated process and Luschka joint stabilizes the anterior part of the cervical vertebral column [28] and it seemed to rescue spontaneous dislocation of the cervical ZJ. Accordingly, the venous-synovial complex seemed to become unnecessary.

Unfortunately, we were not able to identify myofibroblasts, which have a critical role in the production and degeneration of elastic fibers in the ligamentum flavum, near and around the large cervical ZJ using immunohistochemistry [29]. No or few elastic fibers around the ZJ near term suggested that ligament development occurs after birth. In the adult ligament flavum, there are several pathologies such as hypertrophy [30] and disorganization and replacement by collagen fibers [31]. With these pathologies, transforming growth factor-beta production by the vascular endothelium regulates the synthesis of elastic fibers by fibroblasts. Conversely, mechanical stresses induce the production of angiopoietin-like protein by fibroblasts; this protein activates transforming growth factor-beta signaling, which results in ligament hypertrophy and/or degeneration [32]. Nevertheless, to assist the vertebral column extension, the ligament flavum exists not only in the cervical region [2] but also in the lumbar region [33, 34]. Therefore, the cervical-specific venous-synovial complex seemed not to play a role of the mother substrate and/or stimulator of the elastic fiber production.

A cuboidal cell lining was seen in veins as well as synovial recesses in the complex: such synovial cells had been described as cytoplasm-rich cells in fetal synovial tissues of the knee and elbow, but the role is unknown [35-37]. In contrast, as a site of the lymphocyte recruitment, a cuboidal endothelial lining is known well in the postcapillary high endothelial venule of lymph nodes [2]. However, in both of the synovium and veins, the present cuboidal cell lining might simply represent a high activity of proliferation for the growing venous-synovial complex.