Book - Contributions to Embryology Carnegie Institution No.20 part 3

Development of the Cartilaginous Capsule of the Ear

When the present study was undertaken the writer's interest concerned more particularly the process of conversion of the periotic reticular tissue into the walled off spaces that constitute the scala tympani, the scala vestibuli, and vestibular cistern. It was soon found, however, that this could not be satisfactorily treated without a consideration of the earlier history of this tissue and its relation to the surrounding cartilaginous capsule. Therefore, a preliminarj' survey was made of the earlier histogenetic processes of all the mesenchymal elements of the inner ear. The character of these processes will form the subject-matter of the first part of this paper. In brief, they include: (1) the original condensation of the mesenchyme around the otic vesicle; (2) the subseciuent differentiation of the condensed mesenchyme into precartilage on the one hand and periotic reticular tissue on the- other; (3) the differentiation of true cartilage and its manner of growth and alteration in form. After considering these, we shall be prepared in the second part of this paper to take up the alterations in the periotic reticular tissue that lead to the formation of the periotic spaces.

Condensation of the Periotic Mesenchyme

If one looks at the otic vesicle in a human embryo from 4 to 5 mm. long, just as the endolymphatic appendage is becoming constricted off from the remainder of the vesicle, it will be found that the mesodermal tissue surrounding it is about the same in its appearance as that in other regions. There is the brain-wall, the otic vesicle, the ganglion mass connecting them, a few blood-vessels, and the ectoderm; otherwise there is to be seen only a more or less uniform mesenchymal syncytium lying between these structures. Close against the vesicle the nuclei are perhaps a Uttle more numerous. This can be seen in figure 5, which is taken from an embryo 4 mm. long (Carnegie Collection, No. 588). The section passes through the otic vesicle in its longest diameter and shows dorsally the endolymphatic appendage as it appears at this time. Lateral to the otic vesicle is the primary head-vein. A network of capillary vessels is spreading over the brain- wall, not extending quite to the ventral median line. Along the median margin of this sheet of capillaries there forms a larger channel which gradually separates itself from the capillaries and takes part in the formation of the basilar artery, as has been described in the chick and pig by Sabin (1917). The mesenchymatous tissue is denser in some regions than in others. The nuclei are quite sparse ventral to the brain-wall near the median line, becoming perceptibly more numerous as we approach the earvesicle. This increase in the number of nuclei in the neighborhood of the vesicle marks the beginning of the mesodermal condensation that is to form the otic capsule. It is not yet possible, however, to outline a definite layer of these nuclei.

When embryos are examined that are a little older than this it is found that a condensation of the mesoderm around the otic vesicle can be clearly recognized. Such a stage is shown in figure 6, which is from a photograph of a section of a human embryo mm. long (Carnegie Collection, No. 721). Under low magnifications it is apparent that the mesoderm in the region of the vesicle is denser than the adjoining mesoderm, and particularly so on the lateral and ventral surfaces of the vesicle. The condensation of the mesoderm is also beginning on the median surface of the vesicle, but the process there is somewhat slower. The endolymphatic appendage, however, is free from any surrounding condensation ; the mesoderm appears to be unaffected by its presence. The section in figure 6 jxisses transverse to the long axis of the vesicle. A small portion of the brain-wall is shown that is slightly retracted from the surrounding mesenchyme. The area of condensed tissue surrounding the vesicle is thick enough to extend from the surface of the vesicle to about half the distance from the vesicle to the ectoderm.

When analyzed under higher magnifications, it is found that the compact appearance around the vesicle is due to several factors. As compared with the mesenchymal syncytium of the adjoining parts, the nuclei here are slightly larger, are more numerous, and are closer together. The intervening protoplasmic syncitium is also denser and possesses wider trabeculse, with correspondingly smaller spaces between them. This condensed tissue abuts, on the one hand, directly against the epithelial wall of the vesicle and forms a limiting membrane, as can be seen in places where the epithelium is retracted through shrinkage changes. On the other hand, it is directly continuous with the general mesenchymal syncytium, the transition between the two, however, being quite abrupt, as can be seen on careful scrutiny.

In embryos between 11 and 13 mm. long, which is just before the first semicircular duct is separated off from the main labyrinth by the apposition and absorption of the intervening labyrinthine wall, the condensation of the mesoderm has advanced in thickness and extent so that it forms a nearly complete capsule for the epitheUal labyrinth. Such a stage is shown in figure 7, which is taken from a human embryo 11 mm. long (Carnegie Collection, No. 353). This capsule encasing the labyrinth is thicker and denser on the lateral and ventral surfaces of the labyrinth, including the ventral pouch that is to form the cochlea. It remains incomplete on the median surface in the region of the nerve terminations. This latter space is occupied by the rootlets of the acoustic nerve-complex which bridge the short distance between labyrinth and brain and which are invested by a rich plexus of blood-vessels. It is this area that eventually becomes the internal auditory meatus. Slightly more caudal, near the glossopharyngeal nerve, can also be made out a deficient portion of the capsule that corresponds to the fenestra cochleiE (rotunda) and the aquseductus cochleae. A third opening through the capsule is brought about by the endolymphatic appendage. This does not become encased by the capsule, but emerges dorsally to lie between the brain membranes and the skull. At first this latter opening is one in common with the internal auditory meatus. It very soon becomes separated off by the growth of the condensed tissue around the neck of the endolymphatic appendage. In figure 7 the section passes through the long axis of the membranous labyrinth. Only the vestibular portion is shown with the endolymphatic appendage opening out of it. The section passes transverse to the thickened margins of the pouches that are to form the superior and lateral semicircular ducts.

Thus at this time there is completely formed a condensed area of embryonic connective tissue surrounding the labyrinth that corresponds closely in form to the cartilaginous capsule into which it is about to be converted. On examining it under higher magnification there is found very little, aside from the condensation, that distinguishes it as yet from ordinary embryonic connective tissue. The condensed appearance is due to several factors. In the first place, the nuclei are more numerous in a given area. They also tend to be larger and rounder. Furthermore, the protoplasmic syncytium between the nuclei is denser, consisting of more numerous and more branched trabecular. In an embryo 16 mm. long, which had been stained with iron hematoxylin and erythrosin (Carnegie Collection, No. 406) the trabecula; between the nuclei appear granular. This appearance is due to the presence of minute nodes that are found along the trabecular and which are stained deeply by the erythrosin, and add to the density of the tissue. Similar nodes are found in the same embryo in the ordinary mesenchyme in that neighborhood, but are less numerous. This condensed tissue differs in one respect quite definitely from ordinary mesenchyme, in that it is almost devoid of blood-vessels, excepting along its margins. To all appearances it abuts, as in younger specimens, directly against the epithelial wall of the labyrinth.

Differentiation of Precartilage

The histogenetic changes which mark the beginning of the conversion of the condensed mesenchyme into a cartilage-like tissue make their first appearance just after the separation of the semicircular ducts from the main vestibular pouch. This occurs in embryos about 14 mm. long. In embryos about 30 mm. long the otic capsule has the appearance and gives the tinctorial reactions of true cartilage. Thus, in embryos between 14 mm. and 30 mm. long, the otic capsule consists of a tissue that is intermediate between a condensed embryonic connective tissue and cartilage, and this intermediate form is known as precartilage.

The apjiearance of the otic capsule just at the time the canals are forming is shown in figure 8, which is from an embryo 15 mm. long (Carnegie Collection, No. 719). The section jiasses horizontally through the labyrinth. A portion cf the utricle is shown at the bottom of the photograph, and detached from it, above, is the superior semicircular duct. A streak extending from the duct to the utricle still persists. This streak represents the wall of the labyrinth that formerly occupied this place and is now absorbed close up to the inner margin of the duct. Surrounding the cajjsule is a plexus of blood-vessels.

On examination under higher magnifications it is found that the tissue forming the capsule at this time differs very little from the condensed mesenchyme which we have seen in the younger stages. The most noticeable difference is that the nuclei are beginning to stand more apart from each other. This can be seen by comparing figures 7 and 8. In the former the section is lO/x thick, in the latter the section is 40^ thick. In spite of being four times thicker, the section of the older specimen shows only about the same number of nuclei that are seen in the thinner and younger specimen in figure 7.

Between the nuclei there are numerous branching slender processes. The spaces between the processes are not as clear as the spaces in the adjoining subcutaneous connective tissue, but contain a homogeneous substance that stains very slightly with such a dj'e as alum cochineal. The accumulation of this substance is doubtless related to the spreading ajnirt of the nuclei and to the alteration in the branching processes that begins to show at this time. In certain regions the processes between the nuclei become less branched. Larger ones become more prominent and the smaller ones begin to disappear. A common arrangement is to find two or more larger processes uniting to form a loop at the side or at one or l)oth ends of the nucleus. This feature is characteristic of precartilage. There is very little tendency as yet to an accumulation of denser protoplasm around the nuclei.

In embryos cabout 16 and 17 mm. long the optic capsule takes on a definite precartilaginous character. This stage is shown in figure 9, which is from a photograph of an embryo between 17 and 18 mm. long (Carnegie Collection, Xo. 144). The embryo is listed in the collection as 14 mm. long, which is its measurement on the slide. Instead of this we use here its estimated formahn measurement, so as to conform to the other embryos, whose measurements are all given as in formalin. The section passes sagittally through the labyrinth. Above is shown the posterior semicircular duct and just below the center is shown the caudal end of the lateral semicircular duct, at the point where it widens out to join the utricle. By this time the differentiation of the tissue has advanced far enough so that one can properly speak of an otic capsule that is readily distinguished from any other condensed connective tissue. The outlines of the capsule are everywhere distinct. It fuses in part with the cartilaginous skull and it is continuous with the stapes. Embedded in it is the epithelial labyrinth together with its ganglionated nerves. The capsule envelops them entirely, except at the nerve entrance which is to form the internal auditory meatus, also at a point in the region of the jugular fossa that is to become the fenestra cochleae and at the opening through which the endolymphatic appendage emerges.

On comparing figure 9 with figure 8 it will be seen that in addition to an actual increase in size the otic capsule is less uniform in appearance at this older stage. There are areas of denser tissue, or, rather, areas of more deeply staining tissue, which extend as streaks through the capsule inclosing other areas of less deeply staining tissue. The areas of less deeply staining tissue are in the immediate neighborhood of the semicircular ducts, completely encircling them and abutting directly against the epithelial wall of the ducts, as in the previous stage.

On examination under high magnification we find that the tissue forming the otic capsule at this time (embryos 17 mm. long) has for the greater part been transformed into precartilage. Precartilage, as seen in fixed material that has been sectioned and stained by the usual methods, differs from condensed mesenchyme chiefly in the alteration in the network of branching processes that extend between the nuclei. In condensed mesenchyme these appear as a syncytium of delicate refractile processes. In precartilage some of these become more sharply marked and linear, and are looped together so as to inclose an irregular space near each nucleus; the others become very finely subdivided and eventually disappear. \Miile these latter processes are disappearing the area in which they he takes on a homogeneous appearance. It does not take the stain, but it is more opaque than the inclosed spaces around the nuclei. Thus, instead of a syncytium the precartilage tissue gives the appearance of cell-islands separated from each other by a homogeneous matrix.

Regarding the exact structure of this slightly opaque substance our material does not suffice to warrant an opinion. This question must be approached by special methods. I may add, however, that remnants of fibrillar processes are found embedded in this substance for some little time after the walling-off . of the encapsulated spaces or cell-islands. Each cell-island consists of a nucleus encapsulated by a clear space that varies in size and shape and whose contour seems to be formed by the persisting processes of the original syncytium. At first the nucleus is accompanied by very little condensed protoplasm, but this gradually accumulates after the formation of the encapsulated spaces and constitutes a cellbody of endoplasm. The nuclei continue to divide after the encapsulation and they can be seen in all stages of the process. The space shares in the subdivision and for a time each daughter nucleus inherits its own share of the space. The encapsulated spaces, in an embryo 17 mm. long (Carnegie Collection, No. 576), which had been stained deeply with hematoxylin and eosin, contained a homogeneous substance that was tinged with eosin. The substance was collected around the nucleus and filled more than half of the space of the capsule; but clearly it was not protoplasm and was not to be confused with the endoplasmic cell-body which forms later. None of this substance was found in the matrix surrounding the capsules.

The embrj'os in the Carnegie Collection that, on account of the stain that was used and the thinness of the sections, show particularly well the process of the differentiation of the encapsulated spaces are as follows: No. 576, 17 mm.; No. 409, 16 mm.; No. 296, 17 mm.; No. 409, 18 mm.; No. 455, 24 mm.; and No. 453, 23 mm. The order in which they are given indicates their relative development. In all of them areas are found showing different stages in the differentiation. On comjiaring them one could come eciually well to two different conclusions regarding the encapsulation of the nuclei and the differentiation of the matrix. One could either say that the mesenchymal syncytium during the precartilage period undergoes a fusion into a semi-solid, homogeneous, slightly opaque mass in which the fibrils disappear and which forms the precartilaginous matrix, while at the same time selected spaces of the original syncytium develop a sharp margin and become encapsulated, each containing its own nucleus, or, one could say that the substance comjjosing the matrix is deposited in the meshes of the syncytium, replacing most of the fibrils and obUterating the spaces except those selected ones that are inclosed by persistent processes and are encapsulated with an adjoining nucleus. One can not, however, see much evidence for considering the encapsulated spaces as of vacuole formation. They are certainly not vacuoles of the endoplasm, for the endoplasm does not make its appearance until after the spaces have taken on their characteristic form.

Differentiation of Cartilage

The transition from precartilage to cartilage is a gradual differentiation that takes place in the otic capsule of embryos between 25 and 30 mm. long. If one examines an embryo 30 mm. long, such as shown in figure 11, it will be seen on comparing it with younger stages that the main capsular mass has undergone a distinct maturation. This transition is marked by a considerable increase in the amount of matrix combined with a more complete encapsulation of the nuclei, or cartilage cells as we may now call them. As the matrix increases in amount it also changes in its chemical composition, so that it is now possible to stain it differentially.

This tinctorial reaction makes an arbitrary point at which it may be said that precartilage becomes cartilage.

All parts of the capsule do not take part in this process equally. It has already been mentioned that during the period of differentiation of the precartilage the tissue of the otic capsule loses its homogeneous character and some areas of it begin to appear more dense than others. Immediately surrounding the semicircular ducts is quite a wide area of precartilage that appears less dense, which in turn is inclosed b}' the main precartilaginous mass of the capsule whose nuclei give it the appearance of greater density. This can be seen verj' well in figure 10. When we come to embryos between 26 and 30 mm. long this contrast between the two varieties of precartilage becomes more sharply defined, though the relative compactness of the arrangement of the nuclei becomes reversed. The semicircular ducts are then everywhere encircled by an area of temporary precartilage that differs from the rest of the capsule and which is not to become true cartilage, but is to be hollowed out to form the cartilaginous canals. This process of hollowing out the cartilaginous spaces and replacing with reticular connective tissue the precartilage that originally filled them forms a very interesting feature in the development of the otic capsule, to which we will refer later.

The difference between temporary precartilage and true cartilage is shown clearly in figure 11. This section passes transversely through the lateral semicircular canal of an embryo 30 mm. long (Carnegie Collection, Xo. 86). An area of temporary precartilage sin-rounds the epithelial duct, forming a dark circular field outside of which is the more permanent capsular mass. Examination under higher powers shows that the temporary precartilage differs from the main mass in that the nuclei are arranged somewhat concentrically, and there is less space between them than exists in the latter, which is the reason for its darker appearance. Furthermore, whereas the temporary precartilage around the semicircular ducts retains the general histological features that were seen in the ,younger stages, the main capsular mass has matured into well-defined cartilage. A specimen of about this same age is shown in figure 13 (Carnegie Collection, No. 199, 35 mm. long). This specimen was stained only in hematoxylin, which emphasizes the matrix. In such a preparation the cartilaginous matrix is stained intensely blue, whereas the temporary precartilage around the semicircular ducts takes the stain only in its nuclei. The reverse picture is shown in figure 12, where the tissues show an intense nuclear stain. This is taken from an embryo of about the same age as that shown in figure 13. Here, on account of the nuclei and the intervening dense protoplasm, the temporary precartilage forms a dark mass around the semicircular duct. Figures 12 and 13 are like a positive and negative and approximately indicate the outlines of the eventual cartilaginous canal. The area of temporary precartilage graduallyretracts towards the border of the more permanent cartilage, as we shall see in the later stages, and as it does so the space becomes occupied by a reticulum of connective tissue.

In passing from embryos 30 mm. long to older stages, such as shown in figures 12, 13, and 14, the tissues show some advance in the degree of their maturation. Their intense stain-reaction causes the area of temporary precartilage to stand out very conspicuously. On examining under higher powers the section shown in figure 12 (Carnegie Collection, No. 972, 37 mm. long), it is seen that the nuclei in the precartilage area are somewhat more numerous and are more commonly arranged than in the same area in figure 11. The darker appearance as contrasted with the surrounding cartilage is also due partly to the fact that the compact mass of internuclear protoplasm is distinctly tinged by the acid stains, whereas in the surrounding permanent cartilage the matrix is nearlj- devoid of any color, having been decolorized by the differential stain.

In addition to the staining reaction there is now a marked difference in structure between the more permanent cartilage and the temporary precartilage. The latter retains its precartilaginous character. Its more peripheral cells show a slight tendency to capsule-formation. A common form among these is an oblong nucleus with thickened elongated processes at the four corners, resembling the pronged egg-case of the shark, the spaces between the processes on each side of the nucleus being parts of the incomplete capsular space. These cells are arranged in circular lines parallel with the circumference of the canal. The transition into true cartilage is rather abrupt, and on advancing into this region one meets with a characteristic matrix, embedded in which are the completely encapsulated nuclei. The temporary precartilage in its more central layers, near the reticulum, does not show any tendency towards encapsulation. Its nuclei are arranged in concentric layers with a small amount of compact protoplasm between them, resembling an early stage of fibrous connective tissue.

A layer of blood-vessels marks the junction of the temporary precartilage with the reticulum surrounding the semicircular duct. This reticulum appears lighter than the surrounding precartilage because of the free spaces between its slender trabecular. Furthermore, the nuclei are not quite so numerous and are more irregularly arranged. The reticulum does not advance very rapidly in its development, and it is not until we come to embryos between 40 and 50 mm. long that we meet with an extensive reticulum. The development of this reticulum will be described after we have taken up some of the subsequent changes in the cartilage.