Development pattern of tracheal cartilage in human embryos

Abstract Introduction Congenital tracheal anomalies are associated with high morbidity and mortality. The etiology of congenital tracheal anomalies is not well understood, but often attributed to malformed tracheal cartilage. The development of tracheal cartilage has not been described in detail. In this study, we aimed to investigate the development pattern and timing of normal tracheal cartilage to better understand the etiology of tracheal anomalies. Materials and methods The development of tracheal cartilage was examined by studying the trachea in histological sections of 14 healthy human embryos from the Carnegie collection. Two specimens for Carnegie Stages 17–23 (42–60 days of embryological development) were studied. Results At Carnegie Stages 17–19 (42–51 days), a continuous mesenchymal condensation was observed ventral to the tracheal lumen. At Stages 20 and 21 (51–54 days), this pre‐tracheal mesenchyme showed sites of increased condensation indicative of future tracheal rings. Furthermore, growth centers were identified both proximally and distally in the trachea. Characteristic horseshoe shaped tracheal rings were apparent at Carnegie Stages 22 and 23 (54–60 days). Conclusions In human embryos, tracheal rings arise from growth centers in the ventral mesenchyme at approximately 51–54 days of embryological development. The observation of proximal and distal growth centers suggests a centripetal growth gradient, potentially contributing to occurrence of complete tracheal ring deformity (CTRD). Although this study shows new insights on tracheal cartilage development, the exact origin of congenital tracheal defects has yet to be elucidated.

The trachea is composed of 14-21 C-shaped hyaline cartilage rings connected by annular ligaments (Kamel, Lau, & Stringer, 2009;Munguía-Canales et al., 2011). The trachealis muscle forms the dorsal side of the trachea. Prior to embryonic development of the trachea, the primitive foregut is formed through cranial and lateral folding of the endodermal germ layer at approximately 3 weeks of embryonic development (Carnegie Stage 9, 19-21 days). The development of the tracheal lumen subsequently starts when the respiratory diverticulum is formed in the ventromedial part of the foregut at 26-30 days of development (Carnegie Stage 12). The respiratory diverticulum grows in a caudal direction, giving shape to the future trachea. At 28-32 days, the ventrally situated trachea separates from the dorsally situated esophagus and the respiratory diverticulum bifurcates into two primary lung buds (Carnegie Stage 13). The tracheal rings and lung mesenchyme derive from the mesodermal germ layer, as the endoderm only forms the epithelial lining of the respiratory tree. In embryological research, the mesenchymal precursors of tracheal rings have been described to some extent, but the development pattern and precise timing of tracheal cartilage remains unknown (Harjeet, Sahni, & Jit, 2004).
In this study, we aimed to investigate the embryonic development pattern and timing of normal tracheal cartilage as a first step to better understand the etiology of tracheal anomalies.

| MATERIALS AND METHODS
Histological transversal sections of 14 healthy human embryos from the Carnegie collection were studied from the larynx to the main bronchi. The Carnegie collection is a human embryo collection assembled in the beginning of the 20th century by Franklin Mall of the Department of Embryology at the Carnegie Institution for Science (Mall & Meyer, 1921). This collection is considered a reference work for embryological research and its name is linked to the stages of embryonic development. The developmental age of the embryos used in this study ranged from 42 to 60 days, corresponding to Carnegie Stages 17-23. For each Carnegie stage, histological sections of two embryos were available for examination. The characteristics of these embryos (e.g., origin, year of acquisition, crown rump length, sex, fixation medium, staining, and number of slides per embryo) have been described elsewhere (de Bakker et al., 2016;de Bakker, de Bakker, Soerdjbalie-Maikoe, & Dikkers, 2018). Carnegie collection specimen numbers used for this research are 6,521, 6,520, 6,524, 4,430, 2,114, 8,965, 462, 2,025, 7,254, 4,090, 895, and 950. These specimens were examined earlier during development of the interactive threedimensional (3D) atlas of the embryo (de Bakker et al., 2016). The 3D reconstructions of the trachea were made by manually selecting the thyroid cartilage, cricoid cartilage, tracheal rings, and bronchial rings in histological sections using Amira software (Thermo Fisher Scientific, Waltham, MA). If possible, the number of (future) tracheal rings was counted. Furthermore, the length of the trachea was calculated in the mid-sagittal plane by measuring the distance between the cranial margin of the first tracheal ring (in Carnegie Stages 17-19, as no cricoid cartilage is discernable at these stages) or caudal border of the cricoid cartilage (in Carnegie stages 20 and up), and the carina.

| RESULTS
Histological sections were of appropriate quality to accurately assess the trachea and potential tracheal rings in all embryos except for one In Carnegie Stages 17, 18, and 19 (42-51 days), average tracheal length was 1.2, 1.6, and 2.0 mm, respectively. A continuous mesenchymal condensation was seen ventral to the tracheal lumen (see Figure 1a). This mesenchymal condensation was C-shaped, as no condensation was seen on the dorsal side. The uninterrupted pattern did not reveal the site of future tracheal rings.
In Carnegie Stages 20 and 21 (51-54 days), average tracheal length was 2.4 mm and 2.5 mm, respectively. The pattern of the ventral mesenchymal condensation was segmented over the entire length of the trachea, with an alternating increase and decrease of the intensity of the mesenchymal condensation. The sites of increased mesenchymal condensation (i.e., precartilaginous condensations) were considered to be indicative of future tracheal rings. Furthermore, rounded cartilaginous nodules (growth centers) could be identified ventromedially in these sites of pre-cartilaginous condensations, ventral to the tracheal lumen (see Figure 1b). However, these cartilaginous nodules were seen only proximal and distal in the trachea. The In human embryos, (mesenchymal) condensation indicative of future tracheal cartilage was first described by Grosser (1912) and confirmed by Harjeet et al. (2004). This most recent publication stated tracheal cartilage development in four embryonic specimens with a crown rump lengths (CRL) of 11.5, 12.5, 22, and 30 mm (corresponding to Carnegie Stages 17, 18, 22, and 23, respectively).
No growth centers were identified in that study. At 22 mm CRL, mesenchymal condensations were more pronounced cranially than caudally, suggesting a craniocaudal growth gradient. Interestingly, this is in contrast to our findings of proximal and distal growth centers suggesting a centripetal growth gradient.
In experimental animal studies, tracheal cartilage development has been described in accordance with our findings ( Congenital tracheal anomalies concern a spectrum of various tracheal disorders. Some disorders can be explained by extra-tracheal factors, such as congenital vascular abnormalities causing tracheal compression (e.g., pulmonary artery sling, double aortic arch, right aberrant subclavian artery) (Lodeweges et al., 2019). In this group of tracheal abnormalities, it is reasonable to consider the tracheal  (Jacobs & Que, 2013). How tracheomalacia is linked to tracheoesophageal fistula has yet to be elucidated.
The most prevalent cause of congenital tracheal stenosis is complete tracheal ring deformity (CTRD) (Herrera et al., 2007). In CTRD, the cartilage is completely circular instead of horseshoe-shaped and the trachealis muscle is absent. CTRD mainly occurs at the middle segment of the trachea and is accompanied by a variable length of tracheal stenosis. Sometimes CTRD is an incidental finding without causing symptoms of tracheal stenosis. CTRD is seen more often in patients with associated congenital malformations like Down syndrome, tracheal bronchus, or cardiovascular disorders like pulmonary artery sling (Bravo, Kaul, Rutter, & Elluru, 2006;Rutter, Willging, & Cotton, 2004). In mice, the pathogenesis of CTRD is attributed to disruption of a complex network of tracheal mesenchyme differentiation including Hedgehog and Wnt signaling pathways (Sinner et al., 2019). As it is generally assumed that tracheal cartilage develops over the entire length of the trachea at the same moment, the question why CTRD is regularly seen in the middle segment of the trachea remains unanswered. Based on our observation of proximal and distal growth centers, we hypothesize that growth centers forming tracheal cartilage first appear at the proximal and distal end of the trachea and subsequently appear in succession toward the middle of the trachea.
By taking this longitudinal aspect into account when discussing the contributing factors in CTRD, we reckon that this centripetal growth gradient plays a role in the occurrence of CTRD in the middle segment of the trachea. Here, the combination of the disrupted signaling pathways and a centripetal growth gradient may lead to CTRD as unrestrained growth of the cartilaginous nodules may "fill the gap" of the absent trachealis muscle progenitor cells.
A tracheal bronchus (also called bronchus suis or pig bronchus, as it is normal in grazing animals including pigs, cattle, sheep, and giraffes) is defined as a displaced or supernumerary bronchus originating from the trachea (Frandson, Wilke, & Fails, 2009). The disorder is seen almost exclusively on the right side and occurs in 0.1-3% of the general population (Moreno et al., 2019). Theories on its development