# Microscopical Researches/On the Structure and Growth of the Chorda Dorsalis and Cartilage

SECTION I.
ON THE STRUCTURE AND GROWTH OF THE CHORDA DORSALIS AND CARTILAGE.
1. Chorda Dorsalis.
THE Chorda Dorsalis in the larve of frogs and fishes lies in, or in some instances, under the bodies of the vertebra, and is continued behind the coccyx, through the whole length of the tail. It is inclosed by a firm sheath, and forms a spindle-like, consistent, gelatiniform, transparent cord, which is thickest at the commencement of the tail, and thence gradually diminishes in each direction, both towards the skull and the point of the tail. It cannot well be separated entire in recently killed animals, but is best obtained from them in the form of delicate transverse sections. If the animal be placed in water for twenty-four hours or longer after death, and the tail then severed from the body at their point of junction, the chorda dorsalis may be entirely pressed out, by gently scraping along its course from the point of the tail, or from the head, towards the wound. As this does not succeed if the animal be allowed to let out of water for the same period after death, the easier separableness appears to depend upon a penetration of the water between the chorda dorsalis and its sheath; the firmer connexion of it in the fresh condition, however, only upon a more close contact, or wedging in of the chorda dorsalis, and not upon a vascular connexion, for I do not suppose that it contains any vessels. Microscopically examined, it exhibits, as J. Müller has discovered in fishes, a cellular structure in its interior, surrounded externally by a proportionately thin cortical substance (rinde), which is beset with scattered granules. The interior exactly resembles the parenchymatous cellular tissue of plants. (See plate I, fig. 4.) It is readily seen, especially at the point of contact of three cells, that each one is surrounded by its own proper membrane. The cells vary much in size, being ​usually largest in the centre, and becoming somewhat smaller towards the outside. They have an irregular polyhedral shape, mostly with spherical surfaces, which are sometimes convex towards the outside, sometimes towards the cavity of the cell. Their walls are very thin, colourless, smooth, and almost completely transparent, firm, and slightly extensible. They dissolve readily in caustic potash. The rudiments of the chorda dorsalis in the conical interstices of the vertebrae of cartilaginous fishes are not dissolved by dilute or concentrated acetic acid. The chorda dorsalis of fishes according to J. Müller does not become converted into gelatine after long boiling.
The cells of the chorda dorsalis of frog’s larvee contain in their interior a colourless, homogeneous, transparent fluid, which does not become cloudy at a boiling heat; the slight clouding observed in the chorda dorsalis after boiling, appears to be situated more in the cell-walls, which afterwards appear minutely granulated.
In the larva of Pelobates fuscus another formation occurs, inasmuch as by far the greater proportion of these cells containv a very distinct nucleus. It has the appearance of a somewhat yellowish-coloured small disc, of a roundish oval form, rather smaller than a blood-corpuscle of the frog, and almost as flat. (See plate I, fig. 4a, where it is represented from the chorda dorsalis of Cyprinus erythrophthalmus.) In frog’s larvae the nucleus is nearly twice as large. It has a sharp, dark margin, and appears minutely granulated. In this little disc may be seen one, rarely two, and very seldom three dark, sharply circumscribed spots. It thus entirely resembles, both as a whole as well as in its modifications, the cytoblast of vegetable cells with its nucleolus, and microscopically, cannot at all be distinguished from it. Compare plate I, fig. 4a, with plate I, fig. 1a. But it also corresponds with it in its position in the cell. In very many cells, the vertical wall of which is viewed from above, it may be seen that the nucleus lies close on the inner surface of the wall of the cell, or even embedded in the wall. It appears then, as in plate I, fig. 1 a, only still somewhat flatter. I have not, however, succeeded in observing that a lamella of the cell-wall passes over its internal surface, which is also but rarely seen in plants. If the external minutely granulated cortical substance of the chorda dorsalis of Pelobates fuscus ​ be more accurately examined, it is found that the granules are oval, and furnished with a nucleolus, and that, with the exception of their being only about half as large, they entirely resemble the cell-nuclei. This cortical substance is not sharply separated from the proper tissue of the chorda dorsalis; and as the cells of the latter suddenly diminish very much towards the cortical substance, I think that these granules upon the latter are the cytoblasts of flattened cells which form it. Sometimes, although but indistinctly even with a very favorable light, very fine lines may be perceived in the intermediate spaces between these granules, where the cells come in contact, as in the common tabular (or scaly) epithelium. In the chorda dorsalis of the larva of Rana esculenta, where the nuclei in the cells are not distinct, these nuclei in the cortical substance are not seen; the tabular structure, however, is evident in them. One must be very cautious in denying the presence of the cytoblasts, when they are not immediately recognizable. They may in animals, as in plants, attain such a degree of transparency, as renders them very difficult of observation. Thus, I could not for a long time detect them in the rudiment of the chorda dorsalis, which is found in the conical intermediate spaces of the vertebra, in a large Carp, until on a very clear day they appeared very pale but quite recognizable, and of precisely the form above described. They were somewhat more distinct in the Pike and Cyprinus erythrophthalmus. The delineation, plate I, fig. 4, is taken from the latter. They are however smaller in these fishes than in frog’s larvee.
To return to the larva of Pelobates fuscus. Here the cells of the chorda dorsalis lie so close to each other, that the walls of the two neighbouring cells are in immediate contact. Even when three or more cells are in contact, they are generally so close, that only the contiguous walls are observable. Sometimes, however, in such instances, a small intermediate space remains, which is larger than could be filled up by the unthickened cell-wall; and there is then seen, as in plants, a species (apparent or real?) of intercellular substance, or an intercellular canal. With regard to this latter (intercellular canal), occasionally, though rarely, in such an instance of close contiguity of three cells, upon making a transverse section, the cell-walls are observed sharply bounded, as well towards the cell as externally, ​and between the cells a small triangular interstice is seen, which is filled by a transparent fluid (not by air), or at least by a substance which refracts the light in a different manner from the cell-walls, just as it is represented in plate I, fig. 1c, from the onion.
Young cells, which float free, form within the cells of the chorda dorsalis, as in plants. They are, however, in the larvae of the frog so transparent, that very favorable light and good instruments are required to see them. The number of cells, also, in which new ones are formed in the larvæ is not great, at least in such as are to be had in the latter part of autumn. In the above-mentioned species of Cyprinus, and also in other fishes, they are, however, easy to be seen, and in greater number. Vesicles of very various sizes may be perceived in the cavities of many of these cells, and also in those of the larvæ of the frog, though they are more difficult of observation in the latter; a single one of these vesicles sometimes fills the greater part of the cavity; and occasionally several lie in one cell. (Pl. I, fig. 4, b, b, c.) They are commonly quite round; but not unfrequently two are in contact, and flattened against each other. That they lie free in the cell, follows from the fact, that they may be isolated without rupture. If, for instance, a small portion of the chorda dorsalis be torn into minute pieces, and a thin plate of glass with water be placed upon them, by moving this lightly backwards and forwards a few times, some such isolated vesicles may often be brought into the field of vision. They may then be made to roll about, and thus demonstrate their globular form. I have taken great pains to discover a nucleus in their walls, but without success. The young cells of the chorda dorsalis, also, in the larvae so often mentioned, have often the appearance, so long as they are not isolated, of possessing a nucleus: but one may readily be deceived here, since such a nucleus may belong to a cell lying above or below them. Caution must also be used, not to confound a globular epithelial cell, which may have shipped into the chorda dorsalis in making the transverse section, with the true cells of that structure. I have not as yet been able, with certainty, to observe any nucleus, at least not of the characteristic form, in isolated young cells of the chorda dorsalis. In rare instances, a very small corpuscle, (d, d, of ​the figure,) lay in the inner surface of the young cell. It must remain a question whether the nucleus is really wanting, or whether it is only not visible in consequence of its translucency, or whether these corpuscles are developed into the nucleus. The chorda dorsalis accords with the vegetable cells, at least in this respect, that young cells are formed within the old ones.
With regard to the thickening of the cell walls; these appear to remain always simple (unchanged) in the chorda dorsalis of the larva of the frog. But in the fully developed osseous fishes, in Cyprinus, for example, a thickening is exhibited in those cells which lie near the axis of the conical interspaces of the vertebræ. The cell-cavities always become smaller in consequence of this thickening of the walls. The thickened walls, or the intermediate substance between the cell cavities consist of closely cohering longitudinal fibres, between which very fine transverse fibres are also sometimes seen. The longitudinal fibres run uninterruptedly past several cells; and the primitive membrane of each cell can no longer be distinguished.
To sum up the researches upon the chorda dorsalis in a few words; it may be said to consist of polyhedral cells, which have, in or on the internal surface of their walls, a structure, according in its form and position with the nucleus of the cells of plants, namely, an oval flat disc containing one, two, or more rarely three nucleoli. The cells usually lie in close contact with each other; but sometimes at points where three or more cells meet together, a sort of intercellular substance, or an intercellular passage is seen. Young cells, which are at first round, and float free, are formed within parent cells. Nuclei of the characteristic form, are not distinctly observed in these young cells, but sometimes a small globule lies upon their inner surface. In those cells which undergo further development, the cell-membrane ceases to exist as a distinct structure, and the intermediate substance between the cell cavities consists for the most part of longitudinal fibres.
With the exception of the formation of these fibres, into the origin of which I have not yet examined, and the absence of the nucleus in the young cells, these cells entirely accord with the vegetable cells. It must remain undecided whether the nucleus is really wanting in these young cells, as it is not yet proved to exist in all plants, (for example in many acotyledo​nous plants,) or whether the little corpuscle, which presents itself on the inner surface of some young cells, is the nucleus which grows with the cell, as it is observed to do in some other animal cells; or whether the nucleus in the young cells is invisible in consequence of its translucency, since even fully-developed cells are met with, in which, although certainly present, it is, in consequence of its transparency, barely visible.
2. Cartilage.
The accordance of the structure of cartilage with the tissue of plants is of more importance in reference to animal organization. We have here to do not only with a more widely extended animal tissue, but also with one which, at least, in its subsequent stages of development, contains vessels, and therefore bears more decidedly the character of an animal tissue. The simplest form of cartilage is exhibited in the cartilages of the branchial rays of fishes. If, for example, a branchial ray of Cyprinus erythrophthalmus be loosened from the branchial arch, and the mucous membrane be removed by gentle scraping, the cartilage remaining presents the appearance of a little rod, which diminishes from the point of its insertion on the branchial arch towards its free end, its sides being somewhat compressed, and exhibiting on their margins some blunt prominences. The structure of this cartilage is very simple. At the point it perfectly resembles, in its whole appearance, the parenchymatous cellular tissue of plants. (See pl. I, fig. 5, from the above-mentioned Cyp. eryth.) Little polyhedral cell-cavities with rounded corners are seen lying closely together. The cell-cavities are separated from each other by extremely thin partition walls. The cell-contents are transparent, and a small pale round nucleus (a) may be seen in some cells when in the recent state, in others only after the action of water upon them. The structure of the lateral prominences of the cartilage is similar to that at the point, only that the cells are somewhat extended in length. Advancing from that point towards the middle, or still better from the point towards the root of the branchial ray, the partition walls of the cell-cavities are observed to become gradually thicker ; and the cavities are here somewhat smaller. (Pl. I, fig. 6.) On the thickened cell-walls it may now also be seen ​that the intermediate substance of the cell-cavities is not a simple structure, but one composed of the walls peculiar to the contiguous cells: that is to say, each cell is surrounded with a thick ring, its peculiar wall, the external outline of which is more or less distinct. In the preparation from which the delineation is taken, it was in some parts quite as distinct as the internal. Between two cells these external outlines blend into one line, but separate again when the contact of the cell-walls ceases; there is thus often left between the cell-walls a three or four-cornered intermediate space (c), filled with a kind of intercellular substance. No other structure, no deposition of strata, or distinction between primary cell-membrane and secondary deposit can be observed in the thickened cell-walls. The cell-contents also remain clear after the thickening of the walls. At the base of the branchial ray, it is scarcely possible to distinguish between the different cells-walls, and the cartilage presents the appearance of a homogeneous substance, in which separate small cavities only are seen. (Pl. I, fig. 7.) Around some few only of the cell-cavities, a trace of the peculiar cell-walls may be seen in the form of a ring. This ring is usually somewhat thin, so that the entire intermediate substance of the cell-cavities cannot be formed of the cell-walls; but the intercellular substance, which was so small in quantity in the centre of the branchial ray, here contributes essentially to the formation of the cartilaginous substance, and often completely prevents the immediate contact of the cell-walls. This intercellular substance appears, however, to be homogeneous with that of the cell-walls, and in most situations coalesces with them. The cell-cavities, which are here transparent and without granulous contents, are now the cartilage-corpuscles.
The process of formation of this cartilage is as follows. It consists originally of cells, which lie in very close contact, but every one of which has its special, very thin cell-membrane. This follows, firstly, from the complete accordance in appearance, of cartilage in its earliest stage, with vegetable cellular tissue; secondly, from the presence of the nucleus in the young cells of cartilage, a structure which, as will subsequently be seen, occurs in almost all the cells proved to exist in other tissues ; thirdly, from the fact, that a separation of the cell-walls is often distinctly perceptible in instances where they are ​thickened, These cell-walls lie either in close contact, or have only a trace of intercellular substance between them, or there is sufficient of that material to entirely prevent the contact of the different cells. Their walls, which are originally formed of a very thin membrane, become thickened. The cavities of the cells with thickened walls which are seen in the centre of the branchial ray, are smaller than those of the cells which lie nearer the surface, the walls of which are less dense; but, whether this is produced by a thickening of the cell-wall taking place from without inwards, or whether rather the cells were not smaller in their original formation, is a matter of uncertainty. No deposition of strata, nor any distinction from the primordial cell-membrane, can be recognized in these thickenings of the walls. The condensed cell-walls at length coalesce either with each other, or with the intercellular substance, to form one homogeneous mass, in which only the cell-cavities remain perceptible, presenting the appearance of small distinct excavations filled with a transparent substance; these cell-cavities are the cartilage-corpuscles.
In the foregoing description no error can arise from the great variety in form which the cartilage-corpuscles frequently present; for, on examining the branchial rays of a very large pike, the gradual transition may be traced, from the thin-walled almost globular cells to the most varied forms, in which the remains of the cell-cavities are so much extended in length as to give to the cartilage almost a fibrous appearance.
The same extremely simple process of formation (modified, however, in some important respects) is presented in all cartilages. These modifications, the fundamental type of which is already pointed out in the cartilages of the branchial rays of fishes above described, depend chiefly upon the share relatively contributed by the thickened cell-walls, or the intercellular substance, to form the intermediate substance of the cell-cavities, or cartilage-corpuscles. We have seen that this intermediate substance was formed almost entirely of the thickened cell-walls, with but a minimum amount of intercellular substance, in the centre of the branchial rays of fishes, whilst at their base, that is, in the earliest formed cartilage, the intercellular substance preponderated, and the less dense cell-walls contributed less to the formation of the true substance of the cartilage. ​The walls of the cells appear to contribute little or nothing to the formation of the substance of most of the ossifying cartilages,—those of the higher animals for example.
The cartilages of the branchial arches of the tadpole, like those of the branchial rays of fishes, consist of cells, which are, however, much larger than those of the fish, though smaller than the cells of the chorda dorsalis, with which they have, in every other respect, much similarity. The partition-walls of the cells are thicker than in the chorda dorsalis, but they may still be termed thin when compared with the cell-cavities. (See pl. III, fig. 1, which exhibits branchial cartilage from the young larva of Pelobates fuscus.) The cartilage intended to be used for investigation must be taken quite fresh from the living animal; for the structures become very indistinct if it be allowed to lie in water for any time after death, even though it be entire. After stripping off the mucous membrane, the cellular structure is readily recognized by the aid of the microscope. The cells vary much in size, and are more or less flattened against one another. The wall of each separate cell may be distinctly seen in the majority of instances, and its thickness might even be measured; that we cannot trace it so evidently in the smallest cells is probably referrible to the extreme thinness of their sides. ‘The walls of the cells are for the most part in contact, but intercellular substance may be seen in many situations,and especially where several cells are contiguous. The surface of the cartilage, which is represented on the left and lower margin of the figure, (pl. III, fig. 1,) is formed in the first place of intercellular substance, which, in as much as the cells originate in it, may be called Cytoblastema.
This cartilage may, therefore, be described as consisting of intercellular substance, or cytoblastema, in which great numbers of cells are seen. The cell-contents are generally clear as water ; but in the younger and smaller ones (for example, c,) the contained matter is less pellucid, and somewhat granulous. Each cell contains a spherical granulous nucleus, which lies upon the inner surface of the wall, and which again encloses a nucleolus. The size of the nucleus is not precisely alike in all the cells: it is somewhat larger in the large ones, but its size hears no proportion to the increased bulk of the cell; and again, the smaller cells are not much larger than the nucleus which they contain. ​Nuclei, around which no cells have yet commenced to be developed, may be observed in the cytoblastema between the cells in some situations; for example, a and b. These like-wise contain a nucleolus, and are somewhat less than the nucleiin the smaller cells.
The above observations furnish us with a complete representation of the development of cartilage-cells, and show the accordance of that process with the development of vegetable-cells, inasmuch as they exhibit the simultaneous presence in the cytoblastema both of simple nuclei, and of cells containing a nucleus of similar shape and size upon the inner surface of their walls, and which may be observed in all stages of transition, from such as are scarcely larger than the nucleus they contain, to such as are many times its size. Simple nuclei are first present, developed in the cytoblastema. When these have arrived at a certain size, the cell is formed around and closely encompassing them. The cell gradually expands, whilst the nucleus remains lying on a point of the inner surface of its wall. The nucleus, also, increases somewhat in size, but not in proportion to the expansion of the cell. Now these three hypotheses may be assumed from the above facts; either the cell is first developed, and the nucleus subsequently, or both are developed simultaneously, or the nucleus is first developed, and then the cell around it. The first supposition, that the cells are developed earlier than the nuclei, is not possible, since in that case cells would be found at a certain period of development without nuclei. The simultaneous development of a cell, together with its nucleus, as two distinguishable structures, is equally impossible, for then we should observe a stage of development, at which as yet the cell and nucleus had not reached the size of the ordinary nucleus. In order to explain the above observations, we must, therefore, have recourse to the third supposition, viz. that the nucleus is first developed and then the cell around it.
The form of the young cells depends upon the space allotted them for expansion. They are, therefore, either round or angular, according as the neighbouring cells permit of, or limit their regular expansion. Two or more cells are often developed close together in one intercellular space, and thus compress those already formed, and the intercellular substance on the outside of them; ​this fact explains the common appearance of two or four cells lying together in a group, being separated from one another by thin walls, whilst between such groups and the neighbouring cells we see much more intercellular substance.
The cells at first appear finely granulated, and not so transparent as in the more fully developed condition. The thickening of the cell-membrane takes place simultaneously with its expansion. One of the cells in pl. III, fig. 1, exhibits two nuclei, one of which, like those of all the other cells, has but one nucleolus, the other having two. It may be conjectured, that this second nucleus is destined to the formation of a young cell within the larger one.
In the intercellular substance at e in the same figure (pl. III, fig. 1,) may be seen a small corpuscle, surrounded by a granulous and indistinctly circumscribed mass, the rest of the intercellular substance being smooth and homogeneous. This is, perhaps, a nucleus in the act of formation, the nucleolus of which is already developed; and when the granulous mass surrounding that structure has obtained a defined external boundary, it will form a nucleus. If such be the case, we have here an instance of accordance of the development of the germ itself with the formation of the nucleus of vegetable-cells observed by Schleiden.
On examining the cartilage of the branchial arches of the tadpole in the more completely developed state, (pl. I, fig. 8,) we find the cells generally lying in groups, so that two, three, or four lie close together, separated from other groups by thicker partition walls. The special walls of the individual cells are less distinct, but at several spots where three or more cells are in contact, for example, at a, the separation of the walls may yet be seen, and a trace of intercellular substance is also present; the latter, however, is almost homogeneous with the cell-walls. It may also be observed that the cell-walls are thicker in these situations than they are represented in pl. III, fig. 1. Some parallel lines may be seen at various spots in these condensed cell-walls, and the thickening might, in such instances, be supposed to be really produced by a stratified deposition of the substance upon the internal surface of the cell-wall. But at the same time it must be remembered, that every partition-wall between two cells must consist of two layers, each of which ​corresponds to the wall of the corresponding cell. This appearance of strata, however, is observed only in the thick walls between two groups of cells, and as these groups probably originate by the formation of two or four cells within a parent cell, each half of the partition-wall between two groups must (presuming such to be the mode of their formation) consist again of two layers, the one of which corresponds to the wall of the parent cell, the other to that of the secondary cell, so that each partition-wall of two groups must consist of four layers. Although it does, indeed, appear that even a greater number of layers or strata are present, yet I must at the same time remark, that these observations are by no means sufficiently conclusive for the proof of a fact so important in reference to the process of nutrition, and that I am so far from recarding them as evidence of a stratified deposition of the substance, as not to hold such a thing to be even probable. The appearance is probably an optical deception. As before stated, no distinction was found between primary cell-membrane and secondary thickening in the cartilages of the branchial rays of fishes, but it seemed that the cell-membrane had actually become thickened; neither is there any such distinction to be observed in the branchial cartilages of the tadpole.
If the above described groups be assumed to have had their origin by the formation of secondary cells within a primary parent one, in that case, secondary cells which completely fill the parent one have not been developed in all the primary cells, for isolated cells occur in the branchial cartilages of Pelobates fuscus, which are somewhat larger than the secondary ones, but smaller than the other primary cells, and remarkable also, as will be seen immediately, from their contents.
The cells of the branchial cartilages of the larva of Pelobates fuscus just mentioned, contain within them one or more nuclei. (Pl. I, fig. 8, d.) These nuclei, which may be easily isolated, are either slightly oval, or perfectly globular, more or less granulous and yellowish, and apparently hollow. They contain one or two very distinct, round, dark nucleoli, which lie in their interior either close upon the wall, or very near to it. The nuclei (a portion of them at least) appear to lie free in the cell-cavity, for they may readily be isolated. The above mentioned primary cells of the larva of Pelobates fuscus in ​ which none of these secondary cells, completely filling the parent one have been developed, contain very commonly several such nuclei, and also one or more young cells. Pl. I, fig. 8, ff, represents such young cells from the branchial cartilages of the larva of Rana esculenta. They are round vesicles containing a nucleus identical in form and size with those which lie free, but which is situated upon the internal surface of the wall, and never in the centre of the cell. This nucleus is never wanting in the young cells. The cells, however, vary much in size, some being scarcely larger than the nucleus they contain, others twice or thrice as large: From one to three such young cells, in various stages-of development, are commonly found within the primary one, where they sometimes become flattened from want of space. As the figure represents, most of the secondary cells contain these young ones, and but few of them only simple nuclei (such as have no cell around them), in some of the young cells, indeed, a second somewhat paler nucleus appears. These young cells lie free within the primary cell, and may be isolated in the same manner as was described with regard to those of the chorda dorsalis. They appear in the first instance to be perfectly transparent; but gradually obtain a granulous yellowish aspect, and it is remarkable, that the earliest formation of this yellowish deposit takes place generally if not constantly, in the neighbourhood of the nucleus.
It will thus be seen that these young cells, (fat cells ?) which are formed within the true cartilage-cells, furnish us with a series of observations as regards their development, similar to that observed in the formation of the cartilage-cells themselves: namely, simple nuclei, cells closely encompassing those nuclei, and all the stages of transition up to the largest cells; but never have we met with these young cells without nuclei. So that the same conclusions might be arrived at with respect to the mode of their development, as were before with regard to that of the cartilage-cells, namely, that the nuclei are first formed, and around them the cells, precisely as in plants. The nucleus in these young cells, however, does not appear to increase in growth after the cell has once formed around it. The accordance in form between these and the young cells of vegetables is shown by comparing Plate I, fig. 8, with fig. 2, b.
The nucleus of the true cartilage-cells like that of vegetable​ cells is subsequently absorbed. After the cartilages of the branchial rays of fishes have been exposed to the action of water, it is only in the young cells that the nuclei are visible ; they are much more rarely seen in those cells of which the walls are already very much thickened. In many cells of the branchial cartilages of the tadpole, a small nucleus with a ragged outline may be observed, which is probably the cytoblast of the cell in the act of undergoing absorption. These cytoblasts (nuclei) of the true cartilage-cells always lie in the cell-cavity, even when its wall is thickened, and it 1s impossible to distinguish whether they lie free or are still connected with the cell-wall. A twofold explanation is here possible: either the cytoblast separates from the wall after the formation of the cell-membrane is perfected, and falls free into the cavity (as occurs in plants), and at such period a secondary deposition of substance upon the cell-wall first commences; or the thickening of the wall is due to an actual increase of the original cell-membrane, and in that manner the nucleus is pushed inwards, and may remain in connexion with the wall. If a secondary deposition of substance took place before the nucleus was disengaged from the cell-membrane, that body must be enclosed in the wall, and would not lie in the cell-cavity. As both these explanations are possible, it will be seen that no conclusion can be drawn from the position of the nucleus, as to whether the thickening of the cell-wall be a secondary deposition, or an actual growth of the cell-membrane. Sometimes a cartilage-cell presents more than one nucleus; when in such a case the original nucleus of the cell is absorbed, all those observed are probably the germs of new cells, which have not as yet commenced their development. The same fact is frequently observed in plants. The nuclei in the branchial cartilages of the tadpole have for the most part the same size; some, however, which are probably not as yet perfectly formed, are smaller than others. It also often occurs that a nucleus is seen expanded to three or four times the usual size; such instances might be mistaken for young cells without nuclei, but they may be readily recognized by their general aspect. They are more transparent and delicate, and exhibit one or two nucleoli, which are easily detected; when two are present they are widely separated from one another. According to ​Schleiden, a similar enlargement of the nucleus also occurs in plants, thus affording a remarkable accordance in what seems a very unimportant circumstance. It appears to be a kind of abortion; for I have never yet seen a cell formed around such a nucleus.
The cranial cartilages of the tadpole (Plate I, fig. 9) are distinguished from the branchial by the smaller size of the cell-cavities, and the increased strength of the firm intermediate substance. The walls of the separate cells cannot now be traced, they appear to have coalesced with the intercellular substance, which is present in greater quantity. The cells lie in groups of two or four together, and it is very probable, that in this cartilage, each group is formed of cells, which have been developed in a parent cell; for some may be seen, for example at c, which do not as yet quite fill the original cell. Such an instance, however, is rarely so very distinct as not to admit of a doubt. There is a very striking similarity between the group a, fig. 9, and fig. 3, which represents four young vegetable cells developed in a parent cell, and the thickened walls of which have coalesced with one another and with those of the parent cell, so that the four cavities only remain in an homogeneous substance. That portion of the cell-cavities which is still visible is filled with a granulous yellowish substance, in which lie one or more nuclei, or young cells provided with a nucleus: these remains of the cell-cavities are the cartilage-corpuscles discovered by Purkinje.
The intercellular substance is universally much more prominent in the cartilages of mammalia than it is in those hitherto described, and in them it forms the principal part of the firm mass of the cartilage. There is not, however, any essential difference either between the structure of the several kinds of cartilage of mammalia, or between these and the cartilage of lower animals, the only distinction being that it is a little more difficult to prove the existence of the special walls of the cartilage-cells in the former.
The intercellular substance in some cartilages of mammalia is at first so soft, that the cells fall apart under slight pressure, and float free in the fluid. If, for example, a thin lamella be cut off from the cartilage at the angle of the lower jaw of a fœtal pig of three and a half inches in length (a period when ​the cartilage is about to become, but is not as yet, ossified), and placed under the compressorium, the cells will be seen to lie so closely in it, that the space occupied by them may be estimated at three fourths, and that of the intercellular substance at one fourth of the whole volume. Many of the cells which have become separated by the process of cutting, float already in the fluid; and on slightly compressing the preparation many more become loose, and flow out in streams from the intercellular substance into the surrounding fluid. The intercellular substance is too soft to prevent the separation, but at a subsequent period of development this cannot be effected. According to Meckauer the cartilage-corpuscles may also be isolated by boiling. I once succeeded in crushing one of these young carti- lage-cells while still in connexion with the preparation. The first effect of the compressorium was to produce an extension of breadth; it then suddenly shrank together, whilst a clear fluid streamed out, thus proving the contents of the cell to be fluid and transparent. Now, inasmuch as these cells present in different instances a more or less granulous appearance, it follows that the cells of ossifying cartilage must have a peculiar investing membrane, which is granulous, and thus that they are actual elementary cells, in our sense of the word, and neither mere excavations in the substance, nor perfectly solid corpuscles. The appearance of the cells which float about entirely accords also with this view, for while their contents seem to be clear, the cells look granulated. All of them contain a very beautiful oval or circular, not flattened cell-nucleus, situate upon the internal surface of the wall, and this nucleus encloses one or two very distinct nucleoli; in short, they in every respect accord with the elementary cells of most of the other tissues. By the aid of acetic acid we may also frequently succeed in rendering the cell-walls visible upon a thin lamella of cartilage, and as the cell-contents are at the same time disolved by the acid, it has the additional advantage of bringing the nucleus into view, which is sometimes indistinct in consequence of the granulous nature of the contents. Plate III, fig. 2, exhibits a portion of cartilage so treated with acetic acid; it is taken from the as yet unossified portion of the ilium of an embryo pig of five inches in length. The cell-walls, with their double outlines, may be seen, and both the illuminated and ​dark side in the thickness of the walls distinguished. The delineation, at the same time, proves how important a share is taken by the intercellular substance in the formation of the firm structure of cartilage.
The cartilages of the fœtus do not altogether accord in chemical constitution with those of the adult, since we can obtain from them by boiling but a small quantity of a gelatinous substance, and that only with great difficulty, and they afford no true gelatine (capable of forming a jelly). I boiled some unossified cartilages, consisting of apophyses of the femur and cartilaginous portions of the scapule, taken from several embryo pigs, measuring three and a half inches in length. After twelve hours’ boiling, they entirely crumbled into very small scales, which gave a variegated appearance to water in which they were stirred about, and appeared under the microscope extremely thin and granulous. The fluid, when filtered and evaporated almost to dryness, did not coagulate. Alcohol produced a copious precipitate, which was dried, afterwards dissolved in boiling water, and then evaporated almost to dryness; still no coagulation took place. Alum, however, clouded the fluid, and acetic acid had the same effect, but in a much slighter degree. As the quantity of cartilage made use of in the foregoing experiment was too small, I made a further investigation with cartilage which had already become ossified, from the same embryos, namely, the frontal and parietal bones, scapule, humerus, femur, and some ribs. The unossified parts were removed as cleanly as possible from all the bones. The earthy matter was withdrawn by hydrochloric acid; the cartilages were then washed with water, and boiled for twenty-four hours. Under this process they fell to pieces very slowly, meanwhile numerous little glittering scales appeared in the fluid, which, after being dried, resembled very minute fish-scales, and exhibited a beautiful play of colours. They were, perhaps, the lamellae described by Deutsch, which surround the minute medullary canaliculi. The form of most of the pieces of cartilage remained perfectly recognizable, and was but slightly altered. They looked of a yellowish-white colour, and not at all gelatinous, as substances usually do when about to be transformed into gelatine. The fluid was filtered from these little scales and pieces of cartilage, and then evaporated almost ​to dryness. It did not exhibit any trace of coagulation after standing twenty-four hours. After being dried, it was again dissolved in boiling water, on which occasion, however, a portion remained undissolved. It was, therefore, filtered; the fluid was copiously precipitated by alum, and the precipitate was, for the most part, although not entirely, dissolved, on the addition of alum in excess. Acetic acid likewise rendered the fluid very turbid, and an excess of acid did not entirely remove the cloudiness. It was copiously precipitated by tincture of gall-nuts, and acetic acid removed this precipitate again, leaving a very slight turbidness. (Acetic acid likewise completely dissolves the precipitate obtained from glue by tincture of gall-nuts, therefore glue, when dissolved in acetic acid, will not be precipitated by the tincture.) According to these reactions, the gelatinous substance obtained appears to be chondrin, notwithstanding that it was obtained from ossified cartilage. The question, therefore, arises—does the cartilaginous substance which is connected with earthy matter in the fœtus really yield chondrin instead of the gelatine of bone, or was there much unossified cartilage still contained in what appeared to be ossified, and was that the sole source of the chondrin? The point is, at all events, worthy of renewed investigation. It is surprising that the fœtal cartilages should exhibit so great a resistance to the action of boiling water, and that although they yield a small quantity of a gelatinous substance, they do not afford any which has the property of gelatinizing.
The formative processes of cartilage hitherto described, proceed, as it appears, without the presence of vessels in the structure; such at least is the case in thin cartilages, to which probably the fluid parts of the blood can penetrate from the vessels of the neighbouring tissues. In the branchial rays of the fish, for example, I could not find any space in which vessels could have existed; throughout the structure masses of cartilage and cartilage-corpuscles were to be seen, but no canals which could have been traversed by vessels.
The manner in which ossification proceeds now becomes an interesting object of inquiry. The investigation is best pursued by making very fine sections with a razor, from the half-ossified cartilages of the extremities, vertebræ, or coccyx, of the larva of Pelobates fuscus. The little cartilage-cells, which ​are not enclosed one within another, and are for the most part furnished with a nucleus, are readily recognized in the true cartilaginous substance of the unossified cartilages. I am not prepared to state whether this substance is formed by thickening of the cell-walls, or by the intercellular substance. The earthy matter is first deposited in the true cartilaginous substance. It first appears in the form of isolated, extremely minute granules, by which an indistinct appearance of arched striæ is sometimes produced. At other points, these little granules of earthy matter lie collected together into larger irregular heaps. I do not know whether these little collections are depositions of pure earthy matter which has not as yet united with the cartilage, and therefore merely provisional deposits which subsequently are distributed equally in the cartilaginous substance (which is not probable), or whether this earthy matter is already united with the cartilage, and that the regular aspect which the structure presents when ossified may be accounted for by the gradual union of the earthy matter with it after the same mode. I saw no such deposition of earthy matter in heaps in the incompletely ossified parietal bones of the same larva, but the whole cartilaginous substance contained it equably distributed without any perceptible granules. In both instances, however, when dilute hydrochloric acid is applied to the object under the microscope, the boundary denoting the solution of the earthy matter, and the consequent transparency of the cartilage, may be distinctly seen advancing in the form of a sharply-defined line from the edge of the preparation towards the interior, proving that, in the cartilages first mentioned, there was earthy matter equably united with the substance, in addition to the heaps and isolated granulous deposits. For this boundary line cannot be produced by the mere progressive imbibition of the acid without a solution of the earthy salts; at least neither an unossified cartilage, nor one from which the earthy matter had been previously withdrawn and the acid again washed from it, exhibited the phenomenon of such a line advancing towards the interior. During the early period of ossification, when this line arrives at a cell-cavity, it becomes indented proportionally to the size of the cavity, because it does not come in contact with any earthy matter there; the cell-cavities, in the first instance, being free from earthy salts. The reverse, however, is ​the case in the more completely ossified parts; there the cell-cavity remains behind, forming a dark indentation in the line, which as it advances renders the tissue transparent, and leaves the cavity a black spot, from which dark fibres, similar to those of the corpuscles of bone, issue in a stellated form. Shortly afterwards the fibres disappear, then the corpuscle gradually diminishes, and at last vanishes also, leaving a pale spot. Such an appearance could not be due to an air-bubble in the cell-cavity; for in that case, I think, the course of its exit might be followed. It is probably a more compact mass of earthy matter, which does not become dissolved so quickly as that contained in the substance of the cartilage. After this has become impregnated with earthy matter, the cell-cavities are also filled, and when so filled they are the osseous corpuscles. Similar observations might be instituted on the ossified cartilages of mammalia, in which the identity of osseous and cartilage-corpuscles was rendered more certain by Miescher’s researches. The next question which presents itself concerns the nature of those minute fibres which proceed in a stellated form from the osseous corpuscles. After the earthy matter has been withdrawn the corpuscles may still be seen, though rendered very pale by that process; the fibres, however, are not at all visible, although a formation corresponding to them is certainly present in the cartilaginous substance, and their extraordinary minuteness sufficiently explains the invisibility. The same formation might also exist before ossification, but be invisible from the like cause. As these fibres and the cell-cavities become filled with earthy matter simultaneously, and at a later period than the cartilaginous substance, and since they contain the earthy salts in a more compact and less easily soluble mass, it is probable that they are hollow tubes, that is, canaliculi which proceed from the cell-cavities, spreading out into the cartilaginous substance. According, therefore, to the view which we take respecting the cartilage-corpuscles, according as we consider them to be the cavities of cells, the walls of which have become thickened and blended, not only with one another but with the intercellular substance, so as to form the cartilaginous substance; or as we take them for the entire cells, and the intermediate substance of the cell-cavities as only intercellular substance, so must these tubes ​ be viewed either as canaliculi which penetrate from the cell-cavity into the thickened cell-walls, or as hollow prolongations of the cells into the intercellular substance. In the first case, they might be compared to the porous canals of vegetable cells ; in the second, they would correspond with prolongations of cells, such as we shall often again meet with in the progress of this work. Meanwhile, for an example of those cells which are extended out on all sides into canals, and which I have called stellated cells, the reader is referred to plate II, figs. 8 and 9, where those transformations are delineated from pigment-cells. I decidedly give the preference to the latter explanation of the canaliculi, because they pass through the entire thickness of the firm cartilaginous substance, a fact which, in order to be consistent with the first view, requires for its explanation that the substance between the cell-cavities should be formed of the thickened cell-walls, which is certainly not the case in the cartilages of mammalia, as is seen in plate III, fig. 2. The osseous corpuscles, with their canaliculi, would therefore be the cartilage-cells transformed into stellated cells, and filled with earthy matter. We shall return to this metamorphosis of round into stellated cells when treating of the pigment. The resemblance between stellated pigment-cells and osseous corpuscles is sometimes very striking, as is shown, for example, by the pigment-cell which lies to the extreme right in plate II, fig. 9. The compact bony substance is intercellular substance ; it is, however, probable that the walls of the stellated osseous cells form some, if only a very small part, of it.
When ossification takes place, the earthy matter is first deposited in this intercellular substance, and probably at a subsequent period also in the cell-cavities. The deposition often causes the substance to assume a darkish granulous appearance in the first instance, which it afterwards loses, becoming more equally dark. If we assume, what is extremely probable, that the earthy matter is contained in bones in combination with the cartilaginous substance, in a manner analogous to a chemical union, and not in the form of minutely-divided granules, the mode in which the union with the earthy salts takes place may then be explained in two ways: either the earthy matter combines with a particle of cartilaginous substance in such a manner that each smallest atom receives in the first instance a ​minimum of salts, and gradually more and more, until the whole portion of cartilage obtains its due quantity; or, the earthy matter unites at first with some only of the smallest atoms of the cartilage, combining, however, with these to the full proportion which their capacity of saturation requires ; the remaining atoms then gradually and successively receive their due portion of the salts, so that each atom does not chemically combine with them until it can become completely saturated. The latter explanation, from the analogy with inorganic combinations, and from the above-mentioned granulous appearance which cartilage exhibits when undergoing ossification, appears to me by far the more probable. For, according to the first view, the medullary canaliculi, im the neighbourhood of which the deposition of earthy matter first commences, ought to be surrounded, not by a egranulous appearance, but by a dark shadow which should gradually fade away to a pale edge.
I conceive the formation of the medullary canaliculi in ossifying cartilage to be similar to that of the capillary vessels, which will be examined hereafter. We shall return to them again, as also to the origin of the concentric laminae of bone.
We will now briefly sum up the observations upon cartilage, and refer to the phenomena of vegetable life, which either accord with or are dissimilar to them. Cartilage originates from cells, every one of which has its special, and, in the first instance, very thin wall; precisely like those of vegetables. These cells either lie closely together, and on that account are flattened against one another, like those of plants (see pl. I, figs. 5 and 6), or, there is intercellular substance present, and this again either in so very small a quantity as to be visible only in situations where three or four cells are in contact (see fig. 6, c), or in so much greater quantity, as to prevent the contiguity of the different cell-walls (pl. I, fig. 7; and pl. III, fig. 1.) Most of the cells, at their earliest period of development. (and perhaps constantly) contain a nucleus, that is, a round or oval, and sometimes hollow corpuscle (pl. I, fig.5, a; and pl. III, figs. 1 and 2), which again generally encloses one or two nucleoli. The cartilage-cells originate in the first place by the formation of the nucleus in the cytoblastema, around which the cell is afterwards formed, so that the latter at first closely encompasses the nucleus. The nucleus advances slightly in growth after the ​formation of the cell, but in a much lower proportion. It is subsequently absorbed; frequently, however, not before ossification. This is precisely what occurs in vegetables. The walls of the cartilage-cells become thickened (compare figs. 6 and 7 with fig. 5), which is also the case with many vegetable-cells. No distinction, however, between primary cell-membrane and secondary deposit can be observed in cartilage-cells, and such a deposition in strata as is often distinctly seen in thickened cells of plants cannot be made out here with sufficient certainty. The cell-nucleus in the meantime, when not absorbed, remains lying upon the inside of the thickened wall. An instance of actual thickening of the cell-membrane without a stratified deposit, does not, however, appear to be wanting in plants, e. g. the pollen-tube of Phormium tenax. (See the Introduction.) But it seems, that a thickening of the walls of the cartilage-cells does not take place universally, it does not for instance in the ossifying cartilages; the true cartilage substance may also be formed entirely, or at least chiefly of the intercellular substance. The condensed cell-walls subsequently coalesce with one another, or with the intercellular substance, so that at last only the cell-cavities remain in an homogeneous substance. Whether the walls of those cartilage-cells which do not undergo any thickening become blended with the intercellular substance or not, remains uncertain. An analogous instance of coalescence of the cell-walls is afforded by vegetables, for Schleiden has observed such a blending in the layer of bark which less immediately beneath the cuticle of the Cacti.
The cartilage-cells often contain either simple nuclei (i. e. without cells around them), or young cells with such nuclei. These young cells are formed free within the parent-cell, without vascular connexion. Their nucleus is first formed, and afterwards the cell around it, just as in the true cartilage-cell. This is one of the most important instances of accordance between animal and vegetable cells, for the latter, according to Schleiden, are developed in like manner from the nucleus, and likewise within a parent-cell. (See the Introduction.) We may therefore confidently compare the nucleus of these young cells, as also that of the true cartilage-cell, to the cytoblast of vegetable cells. Their shape and the eccentric position of their nucleus, placed as it is upon the internal surface of the cell-wall, ​also accord with the young cells of plants. Compare plate I, fig. 8, f f, with fig. 2. The form of the nucleus likewise corresponds with that of many vegetable cells. In these young cells of cartilage, it is presented to the observer as a small oval or perfectly spherical corpuscle, having, in many instances, a granulous and somewhat yellowish appearance, and containing one or two nucleoli. (Compare this with the description of the nucleus of vegetable cells in the Introduction.) The nucleus of the cartilage-cell appears to be hollow, a fact which has not been observed with regard to the cytoblast of vegetable cells, and the nucleoli lie close upon, or in the neighbourhood of the internal surface of its wall, whilst, according to Schleiden, they lie deep in the cytoblast of vegetable cells.
The cartilage-cells, when once formed, appear to be endued with the capacity to grow throughout the entire mass of the structure. The case is different with regard to the formation of new cells. This takes place in certain situations only, on the surface of the cartilage, for instance, or between the last formed cells. We have already seen that in the branchial rays of fishes, the least developed cells lay at the point, and lateral margins. The little rod, which the branchial ray represents, does not increase in size by the formation of new cells between the original ones throughout its entire length, but its extension in the longitudinal direction is produced by the development of new cells in the neighbourhood of the point, and it increases in breadth by the same process going on in the neighbourhood of the side walls. It is a familiar fact, that the cylindrical bones grow chiefly upon the surface and at the end of the shaft. The formation of new cartilage-cells usually takes place only in the neighbourhood of the surface which is in contact with the organized substance, (I refer throughout this passage to that period alone, at which the cartilage does not contain any vessels of its own) but it is not exclusively confined to that situation, it may also proceed in the intercellular substance between the last-formed cells.
At the period of ossification, the earthy matter is first deposited in the cell-walls, or in the true cartilage-substance, the ​remains of the cell-cavities also become filled with it at a subsequent period, and at the same time the stellated canaliculi issuing from them make their appearance. The formation of these canaliculi probably takes place by the transformation of round cartilage-cells into a stellated form, after the manner of the pigment-cells at plate II, figs. 8 and 9.
The above detailed investigation of the chorda dorsalis and cartilage, has conducted us to this result,—that the most important phenomena of their structure and development accord with corresponding processes in plants, that some anomalies and differences may indeed still remain unexplained, but that they are not of sufficient importance to disturb the main conclusion, viz. that these tissues originate from cells, which must be considered to correspond in every respect to the elementary cells of vegetables. Thus then are we furnished with the first of the proofs required in the Introduction; that is to say, we have shown with regard to a certain tissue, that it not only originates from cells, but that these cells in the process of their development manifest phenomena analogous to those of the cells of plants. We have now thrown down a grand barrier of separation between the animal and vegetable kingdoms, viz. diversity of structure. We have become acquainted with the signification of the individual parts of the animal tissues as compared with the vegetable cells, and know that cells, cell-membrane, cell-contents, nuclei, and nucleoli in the former are in every respect analogous to the parts having similar names in the cells of plants. We have already observed several modifications both of the nucleus and cell. The former presented itself as a corpuscle having either an oval or circular outline, spherical in figure, or very much flattened, sometimes hollow, and often scarcely perceptible, in consequence of its transparency, but generally granulous and yellowish, and containing in its interior from one to three nucleoli. This nucleus lay within, and fast adhering to the wall of the cell, but never in its centre. The fundamental form of the cell appeared to be that of a round vesicle, but we have also observed the flattening of the cells against one another, the presence of intercellular substance between them in greater or less quantity, and lastly, the thickening of the cell-walls. ​We have seen the generation of cells within cells, and the formation both of the young cells in cartilage, and of the true cartilage-cells themselves, was proved to take place around the nucleus, in the same manner as that described by Schleiden in vegetable cells. The other proof for the accordance of animal and vegetable structure (see Introduction, p. 6) yet remains to be supplied, viz. that most or all animal tissues are developed from cells. If this proof only were furnished, the analogy of such cells to the elementary cells of plants would at once become extremely probable; we may now assert that analogy so much the more firmly, since the cells of two distinct tissues have been proved in detail to correspond with those of plants.