Book - Contributions to Embryology Carnegie Institution No.17
Development of Connective-Tissue Fibers in Tissue Cultures of Chick Embryos
By Margaret Reed Lewis.
Up to the present time the study of fixed and stained preparations of embryos has failed to decide the question of the origin of the connective-tissue fibers. This is partly because the methods used necessarily coagulate and distort the delicate cell processes and also the intercellular substances, and such results readily lend themselves to various interpretations by different investigators.
In the following observations on tissue culture an attempt has been made to study the formation of the connective-tissue fibers directly within the living tissue, and not only within living tissue, but within tissue which has developed in an environment entirely free from fibrin or any substances other than those within the cell which coagulates upon fixation.
The pieces of tissue to be explanted for the tissue cultures were washed through several changes of Locke's solution until such fibrogen as was present had become coagulated within the pieces themselves before they were explanted. The medium thus remained free from fibrin, for no fibrin network was observed in the medium of any preparation. In order to see whether the substance forming fibrin would be dissolved out from the explanted piece and deposited as a fibrin network in the medium of the culture, a few cultures were made in which the explanted pieces of tissue were not carefully washed. No fibrin network was found in these cultures, even after many days. However, in such preparations a deUcate network formed over the growth (not within the growth) upon fixation, showing that some substance had been dissolved out from the explanted piece, which coagulated upon fixation. Although this network did not resemble fibrin network, or the delicate processes from the cells, or even the fibrous tissue itself, all such preparations were discarded in order to avoid any possible chance of error.
By this method it is taken for granted that such connective-tissue fibrils as form in the tissue-culture growths arise from the cells, either as a secretion formed by the cells and deposited in the form of fibrils and fibers, or from the transformation of the cytoplasm of the cell itself. As will be seen later, while there is evidence of a possible secretory activity of these mesenchyme cells, as Renaut (1904) and Renaut and Dubreuil (1906) have claimed, due to the presence of the "grains de segregation," or the so-called vacuoles of Lewis and Lewis (1915), nevertheless, in these tissue cultures, the connective-tissue fibrils formed by a transformation of the cytoplasm of the cells.
Tissue culture is not an entirely satisfactory method for the study of any highly differentiated tissue, owing to the fact that the ceUs which migrate out from the explanted piece and later increase by division attach themselves so closely to the cover-sUp and become spread out in such a thin layer that the differeiitiated structure loses its characteristic appearance. Also, the cells of the new growth have a tendency to migrate away as individual cells instead of developing into a differentiated tissue composed of numerous cells. In all probability the cells do not de-differentiate and become more embrj^onic, as has been claimed by Champy (1913) and others, but simply lose their characteristic differentiated appearance, due to their changed shape and position. This is interestingly shown by a study of smooth muscle-cells (plate 2, tig. 6). Where the cells are attached closely to the cover-slip they no longer contract and the mj-ofibrils appear as irregular bundles composed of numbers of dehcate fibrils (plate 2, fig. 6) . However, where the cell is not so closely attached to the cover-slip it continues to contract, and in this case the myofibrils are arranged into the characteristic fibrils. Taking the possible loss of the characteristic appearance of the differentiated structure into account, the very thin and largely spread-out living cells of the tissue culture furnish an excellent means for the study from day to day of certain structures of the cell.
Just how much differentiation can take place in such cells in these tissue cultures is diflScult to state. Certainly in a few cases, where the mesenchyme growth at 48 hours was composed of quite undifferentiated cells, this growth, when kej)t alive by frequent baths of fresh solution, did develop definite connective-tissue fibrils. Muscle fibers have been observed to become more differentiated; but in the case both of the muscle fibers and connective tissue there is some continuation of function, as the muscle fibers frequently contract, and the connective-tissue growth also occasionally contracts back around the explanted I)iece and later grows out again.
Fibrils did not develop in many of the cultures of connective tissue, owing to the fact that the cells remained spread out as individual cells until the death of the culture. In the few cultures that were kept alive for a sufficient length of time, and in which the connective-tissue fibers did develop, they could be clearly seen and studied in the living preparation from day to day, and their development could be traced from the earliest delicate fibril within the exoplasm of the cell to the more adult fibers, which api)car to be free from the cells.
PREVIOUS WRITINGS ON THE LIVING CONNECTIVE TISSUE.
"Whether the connective-tissue fibers arise within the cells or from an intercellular substance is still an open question. The weight of evidence seems to be in favor of a cellular origin, although certain text-books of histology present the question almost wholly from the intercellular point of view.
There are many reviews of the literature on both sides of the ciuestion (Fleming, 1891; Spuler, 1890; Mall, 1901; Rothig, 1907), and also various textbooks, and since the technique used bj' other investigators is so different from that employed in the following observations no effort will be made to take up in detail the various papers upon the origin of the connective-tissue fibers.
While many observers have studied teased iirejiarations of connective tissue, Boll (1872) was the first to study the development of the connective-tissue fibrils entirely from the living cell. Boll made his cultures by teasing out a few of the cells of the tissue to be studied and placing them in a hanging drop of amniotic fluid. Although he did not obtain any growth of the teased-out cells, he was able to observe the connective-tissue fibrils in connection with the cells throughout the different stages of the development of the connective-tissue fibers, and he became convinced, by this study of the living cells, that the fibrils had their origin within the cells and continued through the exoplasm of one or more cells. Boll studied carefully the following tissues: Arachnoid of chick einV)ryo of 4 to 19 days' incubation. Subcutaneous tissue of chick embryo of 7 to 17 daj's' incubation. Cornea of chick embryo of 4 to 21 days' incubation.
Tendon of chick embryo of 7 to 21 days' incubation. / .q -j«\ He concluded that in all of these tissues the connective-tissue fibers originated^ from the cells In the study of the connective tissue by the tissue-culture method, preparations such as those studied by Boll were used, and also many others, in which the tissue was either teased out, or flattened out, or suspended in a hanging drop of Locke's solution instead of amniotic fluid. Figures similar to those given bj' Boll were frequently observed (plate 1, figs. 2, 4, and 5), and his observations were corroborated. However, in all such preparations it is impossible to ehminate the possibility of fibrin or some other intercellular substance taking part in the formation of the fibrils, and for this reason these observations will not be given below, although they undoubtedly show the connection of the fibrils with the cells.
As no cultures containing fibrin have been studied, nothing can be said at this time in regard to Baitsell's observations (1915), by means of which he shows that certain fibers which resemble the connective-tissue fibers may form in a fibrin clot in the presence of a ])iece of tissue of a chick embryo after various periods of time. In the development of the embryo there can be no question of fibrin playing any part in the formation of the fibrous tissue, since, so far as is known, fibrin is not present in the uninjured tissue. Whether the cells of the embryo possess the power of secreting a substance which may act in the same manner as the injured cell to produce the formation of fibrin, or whether the connective-tissue cells in the developing embryo act directly upon the plasma, are questions which Baitsell docs not discuss. He quotes the following experiments of Loeb, from Adami : "When a drop of uncoagulated lymph is placed between two glass shdes, the mere act of pulling one slide over the other leads to the appearance of fibrils, which grow in length and bulk; which hke those of coimective tissue are not only intracellular, but actually traAerse cell bodies situated in their path; which show themselves first in immediate connection with the cells, the cells as we now hold liberating an enzj-me that determines the modification of the more soluble protein into a precipitated or coagulated modification. But the lines of the precipitation are evidently along the lines of strain." These e\perini?nts of Loeb are in a way comparable to those of BaitseU, except that in Baitsell's experiments the strain is brought about by the shrinkage of the plasma clot. It seems rather difficult to draw any conclusions in regard to the manner of the formation of the connective-tissue fibrils in the embryo from results which are so obviously due to injured cells as are those of Loeb's experiments. However, there is a striking resemblance between the fibrous tissue obtained by Baitsell by means of a modification of the fibrin clot and the fibrous tissue of the embryo.
Baitsell's (1916) paper on wound-healing, in which he finds that very shortly after a wound has been made in the skin of a frog, fibrin fibers, which resemble connective-tissue fibrils, are deposited, and that these fibers persist and take part in the formation of the cutaneous tissue, opens the exceedingly interesting question as to whether what takes place in the process of wound-healing can be in any way comparable to the behavior of normal developing tissue.
So far as can be gathered from Isaacs's (1916) incomplete report of his observations upon the living connective-tissue fibers, his results correspond more or less with those of Loeb — that is, that various strains cause the intercellular substance to form fibrillffi. Just what part the cells play in this formation it is difficult to understand. Isaacs does not say that the cells form an enzyme, as Loeb clauns, but states that the movements of the connective-tissue cells probably effect the distribution of the material through chemical or other action and cause the fibrillated structures of the adult fiber. From Isaacs's brief report it is evident that he had performed numerous experiments with the hving connective tissue, and it is hoped that his complete paper will clear up many points.
Ferguson's (1912) observations upon the living connective-tissue cells in the fins of fish embryos are extremely interesting, since by his method the connectivetissue cells were studied under entu-ely normal conditions. Since there has been some question as to whether fibers actually exist or whether they are merely coagulations of a colloid within the tissue due to abnormal conditions, it is interesting to note that Ferguson describes fibers as well as cells as existing in the / living embryo. He found, by the aid of preparation stained by Bielschowsky's ^ method, that the fibers arise within the cell. Unfortunately he was not able to see the fibers in the embryonic cells of the living embryo and to determine whether they become separated from the cells, or how this takes place. His observations upon the movements of the connective-tissue cells show that the round and stellate cells may move up to and stretch along a fiber as a very thin, long spindle cell, and in a few cases he observed such a spindle cell to become stellate again. Ebeling (1913) has for a period of two years or more kept alive certain of the cultures started by Carrel. The growth of these cultures consists mainly of connective tissue, and Ebeling claims that connective tissue may have a permanent life outside the organism when properly cared for. The method of keeping the culture alive is as follows: The entire culture in its plasma clot is freed from the cover-slip and washed in Locke's solution to remove any waste products and is then cut into fo\ir or more jneces, and each of these pieces is again explanted into a drop of fresh ])lasma. This procedure is carried out every other day; although many of the cultures die, a few survive and grow, and are again exi)lanted as described above. In all i)robability there is no differentiation of the connective tissue into fibrils or fibers, as Ebeling describes the growth as though it consisted of undifferentiated mesenchyme, which is what would be expected in any tissue that proUferates as rapidly as this tissue necessarily must. It would be interesting to see whether, if one of the cultures were kej^t alive without further explanation, it would again differentiate after a certain equiUbrium of prohferation had been reached.
Some workers have claimed that certain substances present in the medium of tissue cultures prevent the growth of the connective tissues. For example, Walton (1914) states that liver extract inhibits the growth of adult mammahan connective tissue in plasma cultures, and Russel (1914) claims that gentian violet, in solution of 1/20,000 in the medium of tissue culture, prevents the growth of connective tissue but not of endotheUum. The reason for this neither writer explains, nor does either state what structure of the cell is affected by the substance so that the cells do not grow out, or whether the medium may simply not attract the cells to migrate and that the cells themselves are uninjured.
Thus a review of the literature on the living connective-tissue cells shows that the study of the living tissue has not presented decided proof as to whether / the connective-tissue fibers arise from the cells or are formed from an intercellular substance. Evidence is presented on both sides, and the question remains as completely at a dead-lock as when the observations were confined to fixed and stained preparations.
OBSERVATIONS IN GENERAL.
A few general observations as to what takes place in the tissue cultures of connective tissue are given here in order to show what factors influence the growth of the fibers.
Cultures from the subcutaneous tissue of chick embryos of various ages were made in the usual manner (W. H. and M. R. Lewis, 1915; M. R. Lewis, 1916). Lewis and Lewis have shown that while the cells in the new growth in tissue cultures are under somewhat abnormal conditions as regards environment and nourishment, nevertheless they are activelj- growing cells which undergo normal division and which grow out as definite types of cells — that is, nerve-cells, musclecells, heart-muscle cells, endoderm of the intestine, epithehal cells of the skin, and connective-tissue cells.
As has been stated, no fibrin is present in the medium, and no substance which coagulates.
The subcutaneous tissue can be removed as a thin, transparent sheath from the skin of chick embryos of 10 days or older. It proved difficult to isolate the subcutaneous tissue from embryos younger than 8 days; and in these embryos a piece of skin or one of the deeper skin fascias or the arachnoid tissue was used for explanation. The connective tissue of embryos less than 8 daj's old is composed of cells without definite fibrils; that of embryos of 11 days and over contains definite bundles of fibers. The new growth from an explanted piece of subcutaneous tissue of enl^ryos of 8, 9, and 10 days proves very satisfactory for the study of the development of the fibrils. The growth can be kept ahve and healthy by frequent baths of fresh Locke's solution, plus 10 per cent bouillon, plus 0.25 per cent dextrose; and fibrils begin to develop in the new growth in from 48 to 72 hours and continue to develop until the growth is about days old or over. Cultures of the subcutaneous tissue from an 11 or 12 day chick embryo also prove very satisfactory for study, for not only is the new growth available for study, but the explanted piece itself is so thin that the cells and fillers can be observed even with the oil-immersion lens.
The fibers in the exjjlanted piece were not observed to grow either in length or bulk, and after a jjeriod of two weeks they remained much the same as when explanted. In no case was a fiber observed to jiass out from an explanted piece over the new growth; such a fiber always remained curled up within the explanted piece.
New fibrils begin to develop in the new growth from an explanted piece of tissue from an 8 to 12 day chick embryo shortly after the new growth is 24 hours old, and definite bundles of fibrils may be developed when the growth is 5 or 6 days old. These fibrils develop more quickly in growths from the older embryos of 10 to 12 days than in those from the younger embryos of 8 to 10 days.
The new growth from the subcutaneous tissue is extremely sensitive and reacts to all sorts of changes in its environment, by contraction. Frequently while a membrane of connective tissue was under observation it would begin to contract from the outer edge of the growth and draw in towards the explanted piece. This contraction might stop at any period or it might continue until the entire new growth had contracted close to the explanted piece. The explanted piece was never observed to contract. The relaxation after such a contraction was exceedingly slow, and frequently a contraction that had taken no longer than 2 to 5 minutes required for relaxation from 1 to 6 hours. In fact, the process did not resemble relaxation, but rather a growing-out again of the new growth. Often, coincident with the contraction, there occurred a rolhng-back of the edge of the growth, and in this case when the cells migrated out again manj' of them became changed in their relative positions. Thus it is evident that a decided strain is present during the development of the fibrils, though there is no fibrin and (so far as can be seen) no substance which coagulates surrounding the cells. Whether this strain or tension (often exhibited by the contraction above described) may in any way influence the separation of the fibrils from the cytoplasm of the cell, it is impossible to state. It was not certain that a pre]xiration which contained well-develoiied fibrils had not contracted during the de\el(>i)nient of the fibrils. However, it can be definitely stated that no substance formcHl into fibrils during contraction, as might have been expected from the exi)(>riments of Loeb and of Isaacs. The new growth, when relaxed after such a contraction, never contained any suddenlj-^ formed fibers or fibrils, and such fibrils as were jiresent were in very much the same state of development as that in which they were before the contraction Untk place. Also, many membranes in which no fibrils ever develojied jiossessed the i)()wer to contract, and did contract more than once.
From these general observations it is evident that the fibers which form in the ti.ssue cultiires must ari.se from the cells; and since the cells are spread out in a thin layer the process of development of the fibers can be ob.servecl in the living cell undisturbed Viy any manipulatioii.
OBSERVATIONS IN DETAIL.
The growth from a piece of chick embryo of 6 to 8 days' incubation is usually in the form of a membrane closely attached to the cover-shp, and is composed of large, flat cells, either connected by numerous cytoplasmic processes (plate 1, fig. 1) or else crowded together so that the delicate processes from cell to cell are lost and a more definite cell-wall appears (plate 2, fig. 10). The growth from older chick embryos may also sometimes have the apj^earance of a membrane, especially where the cells are spread out in a thin layer along the cover-slii). When such a growth is treated with silver-nitrate stain the membrane becomes marked with more or less definite cell-walls, according to the amount of crowding of the cells (plate 2, fig. 10). Such a membrane has been described by Clark (1914), where the connective tissue is stimulated to grow out over a very smooth surface, which Clark interprets as showing that under certain conditions the connective-tissue cells may become transformed into endothelium. While the pattern which appears with the silvernitrate stain is in many ways characteristic of endothelium, still growths from older chick embryos (8 to 10 days) in these tissue cultures exhibited the characteristic activities of connective-tissue cells, and in some cases fibrils were formed within the cytoplasm of the cells (plate 2, fig. 10).
The growth from an 8 to 10 day chick embr3'o usually has the appearance of a reticulum of cells (plate 1, figs. 7 and 8). Some of these cells are of the large, flat, stellate tjpe, having processes on all sides, in which may develop bundles of fibrils, which pass in more than one direction through the ceUs (plate 1, figs. 7, 8, and plate 2, fig. 10); others are cone-shaped — i. e., while the cell-bodj' may have several short processes, most of the cj^toplasm is drawn out into one long process (plate 1, fig. 2, and plate 2, fig. 4). Both the granular and the clear cytoplasm is continued out into the one long process, which i)ractically always extends in the direction from which the cell has migrated, and although in many cases it continues back as a delicate thread, passing as many as twelve or more cells, it has always a protoplasmic end, either free or closely attached to another cell. These long processes usually contain mitochondria and other granules scattered along their length, and never in anj' case have they been observed to change into connective-tissue fibers. In many film preparations" of the subcutaneous tissue studied while alive, such long.dehcate processes have been observed to extend along the side or through the middle of a bundle of fibrils. This is probably due to the fact that, through some stress, the cell has been drawn out into this shape, either from migration or manipulation, and the fibrils are those which were originally in the exoplasm of the cell.
During the beginning migration (1 hour after explantation) of the cells in the explants from older chick embryos (10 to 15 days), when certain of the cells first begin to migrate it is seen that they are drawn out into exceedingly long and delicate processes which ramify in all directions, as though their cj-toplasm had extended a greai Vngth along the fibers of the subcutaneous tissue (plate 1, fig. 3). As the cell continues to migrate towards the peripherj' of the explanted piece or out into the culture medium, these long processes are drawn into the cell, until finallj'
54 de\t:lopment of fowEcnvE-TissrE fibers.
it becomes stellate in form and later divides by mitosis and may again develop long and delicate processes among the cells of the new growth.
From a study of this cell (plate 1, fig. 3) and of the cells shown in plate 1, figure 8, and plate 2, figure 4, it can be seen that in the embryo, where the growth is in all directions rather than in a flat plane (as in tissue cultures) , a section must necessarily cut many of these dehcate processes and cause the appearance of a network of isolated protoplasmic threads between the cells, because the connection of these threads with the cells to which they belong is not shown in the section.
TjTDical spindle-shaped cells never appeared in pure cultures of connective tissue, but always in those which contained muscle-cells; and in everj- instance a tjTiical spindle-cell could be identified as a muscle-cell.
In certain of the explanted pieces spindle-cells were observed, but these appeared to be due to the pull which had been put upon the tissue during manipulation, for frequently parallel bands of fibers extended along these cells.
In some preparations from a 10-day chick embryo the cells were connected by so many dehcate processes that a network of these processes was formed between them, which would have been difficult to identify as cellular in origin had it not been for the fact that during the mitosis of one of these cells all the dehcate network connected with the cell was partly drawn into it, and the space around the cell became free from network (plate 2, fig. 5) . It thus became clear that the protoplasmic network between these cells was not extracellular in origin.
The fibrils appeared first (after 24 hours' growth) as slightly more refractive lines within the cytoplasm of the individual cells (plate 2, figs. 1 and 9). The mitochondria were frequently stretched along these delicate lines; by careful study, however, it was seen that the mitochondria did not take part in the formation of the cellular fibrils, but that even though they stretched for a certain distance along a fibril they later separated from it.
As the growth became older (48 to 72 hours) the cells become more and more densely connected by delicate processes with cells at a distance; and the refractive line of the primitive fibril ai)peared more and more within the cell and became partly gathered into bundles at one point or another (plate 2, figs. 1 and 2). The cellular cytoplasm became separated into an endoplasm — that is, the granular cytoplasm which contains mitochondria, fat, neutral red granules, etc. — which immediately surrounds the nucleus, and an cxoplasm, or the clear, non-granular cytoplasm of the more remote surfaces of the cell (figs. 10 and 13).
The delicate fibrils of the cytoplasm continued from one cell to another, usually through the exoplasm of the cell processes (plate 1, fig. 9 and plate 2, fig. 2) and appeared in the living cell as clear, slightly more refractive lines of exoplasm, extending from one cell to another, and frequently across or through the exoplasm of one or more cells.
As the fibrils develoi)ed from day to day the bundle became more definite and more independent of the cyt()])lasm of the cells, until finallj- it extended as a slender, clear fiber across several cells (plate 2, fig. 3), and except in cases where the individual fibrils can be traced into a cell, the bundle, or fiber itself, appeared quite independent of the cytoplasm of the cells (plate 2, figs. 2 and 3).
No mitosis was observed in cells whose exoplasm was actively developing into fibrils during the time in which the exoplasm contained the fibrils. Mitosis, how^ever, continued, and many cells were seen to undergo mitosis in regions where other cells were forming fibers. So far as can be determined from these observations, the cell may again undergo mitosis after the bundle of fibers has become independent of the cell cj^toplasm. Whether in such cases the cell actually separates itself from that part of its exoplasm which has been differentiated into fibrils or whether it simply divides the undifferentiated cytoplasm and meanwhile remains attached to the differentiated exoi)lasm (or fibers) could not be determined. However, the cells which contributed fibrils to a fiber bundle gradually increased in number and extended over a wider territory, and the bundle became differentiated into a more and more definite fiber (plate 2, figs. 1 and 3).
The study of the living cell, as well as of the fixed preparation, led to the idea that the fibers became more and more separated from the ceUs, although it is quite possible that they may merely continue through the exoplasm and become more definite, on account of the separation of the cells. Certainlj- in no case, in these tissue cultures, did the fiber become so well developed that the ending of the various fibrils which made up the fiber could not be traced into the exoplasm of a cell (plate 2, figs. 2 and 3) . No completely differentiated fiber was observed throughout its development, although in a few instances, where the cultures were kept in a health}^ condition for several weeks, fibers which resembled those of an 18-day chick embryo were developed. It seems probable that the development of these fibers was bj' a continuation of the process described above.
Certain preparations which had been carefully studied during their growth and development were fixed and stained, and from these preparations most of the jihotographs and drawings have been made. A few of the U\dng cells were drawn on successive days, and although it was frequently impossible to determine the exact cell drawn the day before, at least a cell in its near neighborhood was taken.
MITOCHONDRIA AND THEIR RELATION TO THE CONNECTIVETISSUE FIBRILS.
One of the most convincing arguments in favor of the view that the fibrils arise within the cytoplasm of the cells is the fact that freciuently a few mitochondria are seen along a primitive bundle of fibrils (plate 2, fig. 2) and that occasionally a few are found isolated within a well-developed bundle of fibrils in the primitive fiber (plate 2, fig. 3). So far as is known, mitochondria can not exist extracellularly.
The mitochondria of the cells of the growth from a 6-day to 10-day chick embryo are usually of several types; that is, the granular, the short-rod, and the long-thread or filament type. The greatest number are filaments. Mitochondria are scattered throughout the cytoplasm and occasionally along the network of cell processes between the cells, and thej^ may be arranged in a row along a cytojilasmic process (plate l,fig. 6). In a few instances a single filamentous mitochondrium has been observed to lie along the length of such a process (plate 2, fig. 3). A mitochondrium may be stretched along a fibril in such a way that in a fixed preparation it would be difficult to determine whether or not it took part in the formation of the fiber. However, a study of the Uving cell shows that the mitochondria retain all their characteristic activities. They continue to bend, twist, and migrate, with the result that a mitochondrium, even though stretched for a time along a fibril so that it appears to be part of the fibril, very soon bends and later may move awaj'. ^Mitochondria arranged in a row along a cell process do not necessarily remain there, but may migrate into the body of the cell again.
In the older cultures the cell processes are usuallj^ free from mitochondria. In these cultures the mitochondria are more or less centralized around the nucleus — • i. e., within the endoplasm of the cell.
There is present in certain of the cells another structure, which stains in the manner characteristic of mitochondria with the various mitochondrial stains — red with Bensley's anilin-fuchsin methylene green stain (plate 2, fig. 7), black with iron hematoxylin (plate 1, fig. 6), and i)urple with Benda's method. This structure is in the form of a deposit along certain lines of the surface of the cell (plate 1, fig. 6), and is not present in the cell in its earlj'^ development, but appears later along the edge or on the surface of the cell, and in certain cells, although not usually in those of subcutaneous connective tissue, frefiuently seems to be associated with the formation of fibrils. It seems probable that it is this structure rather than mitochondria which ]\Ie\'es (1910) had under observation when he described the formation of the fibrils of the tendon as taking place from the mitochondria after they had become arranged along the surface of the cell. In the stained jn-eparation this structure definitely resembles the mitochondria, and it would hv difficult to determine whether mitochondria take ])art in its formation. IIowev(>r, the living cell shows clearly that the structure is along the surface of the cell and that the mitochondria do not take part in its formation. Also, while the structure is fixed and stained by the same methods which fix and stain the mitochondria, it is not necessarily destroyed by the agents wliicli destroy initoclioiuhia, l)ut may be present in preparations in which the mitochondria have been destroyed. It is very similar to the structure which forms tlie fibril of the muscle-cell (plate 2, fig. 6).
Alislavsky (1913) was alile to differentiate a plasma fibril as well as mitochondria in the kidney tubule cells. He found that while the plasma fibrils stretched entirely across the cells as straight lines, the mitochondria did not pa.ss to the walls of the cells.
In the cultures studied mitochondria did not fuse into strands or become arranged in rows to form the connective-tissue fibrils. In all of these observations, while the mitochondria at times remained caught within a bundle of fibrils, the fibrils themselves originated from the exoplasm of the cell.
OTHER GRANULES AND "GRAINS DE SEGREGATION" OF THE CONNECTIVE-TISSUE CELL.
The connective-tissue cell ordinarily contains very few fat globules, and frequently none at all. When present they are small, round, highly refractive globules, which usualh- lie near tlie nucleus and which stain in the manner characteristic for fat.
In addition to the mitochondria granules in the cells, there are a number of small, round granules, which can be cUstinguished from the granular tv-pe of mitochondria onlj^ by the rapidity of their movements and by certain vital dyes. These granules stain blue with pyrol-blue, purple with brilliant cres3d-blue, and red with neutral red. In the fixed preparation they frequently take certain of the mitochondrial stains, and especially do they take the same purple color as the mitochondria with Benda's stain.
The vacuoles of Lewis and Lewis (1915) correspond more or less with the grains de segregation of Renaut (1904, 1907) and Renaut and Dubrieul (1906), which these observers found to stain with neutral red and which they claimed formed fibrils through a secretory activity of the cell. These bodies are present in the connective-tissue cell, sometimes in large numbers (plate 2, figs. 2 and 3). but so far as could be determined they take no part in the formation of the fibrils.
Only a few growths of cells which could be definiteh' identified as tendoncells took place, and in these growths the formation of the fibrils occurred in a manner somewhat different from that of the formation of the fibrils of the subcutaneous tissue. The tendon-cells were arranged as narrow, elongated cells, more or less parallel, and the fibrils developed as clear lines along the surface of the cells. These delicate lines joined into bundles from one cell to another in markedly' parallel fines (plate 2, fig. 8). In prejiarations stained with MaUory's connective-tissue stain the fibrils stained blue.
A study of the fibrils of the amnion was undertaken in order to see whether the observations of Pcterfi (1914) could be corroborated in tissue cultures. Peterfi observed vacuoles within the epithelial cells of the amnion, and concluded from liis preparations that these vacuoles fused together and became more numerous in the cells of the amnion of chick embryos of from 3 to 5 days' incubation. Accordins to Peterfi, the walls of these vacuoles contain a substance which is different from the remainder of the cytoplasm, and as the walls fuse together they form a network of elastic fibers over the epithelial cells of the amnion of a chick embryo of 7 days' incubation. j\Iy observations on the amnion in tissue cultures did not show this. The epitheUal cells contained varying numbers of vacuoles, most of which stained with neutral red, though a few remained unstained. Fibrils are formed, but, so far as can be determined from these observations, they are formed from the exoplasm of the cell, regardless of the vacuole, in practically the same manner as are the fibrils of the connective-tissue cells (plate 2, fig. 9).
- The connective -tissue fibrils begin to develop in the subcutaneous tissue of chick embryos of from 9 to 10 days' incubation, and appear as well-developed fibers in the subcutaneous tissue of a 12-day chick embryo. The new growth from explanted pieces of subcutaneous tissue from chick embryos of 8 to 10 days' incubation proved the most satisfactory for the study of the connective -tissue fibers.
- The cut fibers which are present in the explanted piece of 11-day to 15-day chick embryo subcutaneous tissue do not grow either in length or bulk in the tissue cultures.
- The new growth of connective tissue is exceedingly sensitive and reacts by a contraction of the cell from the outer edge in towards the explanted piece. This contraction does not cause the formation of fibers in the new growth.
- P'ibers are not present in the 24-hour growth from even 12-day to 15-day chick-embryo tissue, but develop in the cells of the new growth from delicate fibrils in the exoplasm of the cells after 24 hours.
- The fibrils developed as delicate lines of the exoplasm of the cell; they became gathered into bundles which ixassed from cell to cell, and the bundles later passed over or through the exoplasm of several cells as a definite fiber. The fibers never became so adult that the individual fibrils which make up the fiber could not be traced into the cytoplasm of some cell, whether near or distant from the main bodj' of the fiber.
- Although the new growth, when closely attached to the smooth covcrslip, often takes the form of a membrane, and although this membrane exhibits the cell jjattcrn which is characteristic for endothelium when treated with silver nitrate, nevertheless there is no evidence that these cells have become endothelial cells; they still retain the characteristics of connective-tissue cells, and many form fibrils.
- The mitochondria do not take part in the formation either of the fibrils or of the fibers in these cultures of connective tissue.
- There was no evidence that the fibrils are formed by a secretory activity of the grains de segregation (vacuoles) of the connective tissue.
- The fibrils of the epithelial cells of the amnion appeared to form in the same manner as those of the subcutaneous tissue — i. e., from the exoplasm of the cell, and not from the fusion of the walls of the vacuoles.
Adami, J. G., 1908. Principles of patholoE>', Phila. and Lond.. I, 391. Baitsell, G. a., 1915. The origin and structure of a fibrous tissue which appears in living cultures of adult frog tissues. Jour. Exper. Med., Lancaster, Pa., xxi, 453 479, 5 pi. , 1916. The origin and structure of a fibrous tissue formed in wound liealing. .\nat. Rec, x, 175. Boll, Fr., 1872. Uutersuchungen uber den Bau und die Entwicklung der Gewebe. Arch. f. mikr. Anat., Bonn, VIII, 28-68. 1 pi. CuAMPY, C., 1913. Note de biologie cytologique, . . . Le muscle lisi-e. .\rch. de zool. exp^r. et g6n.. Notes et rev., Paris, uii, 42. Clarke, W. C, 1914. Experimental mesothelium. Anat.
Record, Phila.. viii, 95. Ebeling, a. H., 1913. The permanent life of connective tissue outside of the organism. Jour. Exper. Med., Lancaster, Pa., xvii, 273-285, 2 pi. Ferguson, J. S., 1912. The behavior and relations of living connective-tissue cells in the fins of fish embryos, with special reference to the histogenesis of the collaginous or white fibers. Amer. Jour. Anat., Phila., xiii, 129-149. Flemmi.vg, W., 1897. Ucber die Entwicklung des poUa genen Bindegewebe.s-fibrillen bei Amphibien und Sau getieren. Arch. f. -Vnat. u. Entwcklngsgesch., Leipz., 171-190, 2 pi. IsA.\cs, R., 1916. An interpretation of connective tissue and neuTogliar fibrillse. .\nat. Record, Phila., x, 206. Lewis, M. R., and W. H. Lewis, 1915. Mitochondria (and other cytoplasmic structures) in tissue cultures. Amer.
Jour. Anat., Phila., xcii, 339-401. Lewis, M. R., 1916. Sea water as a medium for tissue . cultures. Anat. Record, Phila., x, 287-299.
Mall, F. P.. 1902. On the development of the connective tissues from the connective tissue sync>-tium. Amer. Jour. Anat., Bait., i, 329-365.
Meves, Fr., 1910. Celjer Strukturen in den Zellen des cmbryonalcn Stutzgcwcbes, sowie uber die Entstehung der Bindegewebes-fibrillen, insliesondere denjenigen der Sehne. .\rch. f. mikr. Anat., Bonn, Lxxv. 149-208.
Mislawsky, a. N., 1913. Plasmafibrillen und Chondriokonten in den Staljchenepethelien der Nicre. .\rch. f. Mikr. Anat., 1. .ibt., Bonn, lxxxiii, 361-370, 1 pi.
PfrrERFi, T., 1914. Beitrage zur Histologie des .\mnions und zur Entstehung der fibrillaren Strukturen. Anat. Anz., Jena, xlv, 161-172.
Renact. J., 1904. Sur espfece nouvelle de cellules fixes du tissu conjonctif: les cellules rhagiocrines. Compt. rend. Soci6t* de biol., Paris, lvi, 916-919.
, 1907. Les cellules connectives rhagiocrines. Arch.
d'anat. micr.. Paris, ix, 495-606, 3 pi.
and G. Dlbrecil, 1906. Sur les cellules rhagiocrines hbres du liquide des diverges s^reuscs. Compt. rend. Social* de biol., Paris, lx, 34-37. 126-129.
R5THIG, P., 1907. Entwickelung der clastischen Fasern. .\nat. Hefte, 2. Abt., Wiesb., xvn. 300-3.36.
RrsSELL, D. G., 1914. The effect of gentian \-iolet on protozoa and on tissues growing in \-itro. Jour. Exper. Med., Lancaster, Pa., xx, 545-553, 1 pi.
Spuler, a., 1896. Ueber die Entstehung der Bindegewebsfibrillen und den feineren Bau den Bindegewebszellen. Anat. Hefte, 1. Abt.. Wiesb., vii, 117-160, 6 pi.
Walton, A. J., 1914. Effect of various tissue extracts upon the growth of adult mammalian cells in vitro. Jour. Exper. Med., Lancaster, Pa., xx, 554-572, 5 pi.
Explanation of Plates
1. Photograph of the membrane of connective tissue of 48-hour growth from a tissue culture of a piece of stomach of a 6-day chick embryo. 4 oc. and 4 mm. lens. Zeiss.
2. Photograph of cells and fibers in a film preparation of subcutaneous tissue of an 18-day chick embryo. Os. vap., iron hem. 4 oc. oil-imm. lens.
3. Camera-lucida drawing of a living cell from a culture of subcutaneous tissue of 14-day chick embryo, 3-hour growth. 6 oc. 4 mm. lens.
4. 5. Camera-lucida drawings of living cells in a hanging-drop preparation, after Bell, of subcutaneous tissue from a 14-day chick embryo in Locke's solution. 4 oc. 4 mm. lens.
6. Retouched photograph of 4S-hour growth from uraclmoid tissue of 7-day chick embiyo. Ot. vapor, iron hem., 4 oc. oil-imm. lens.
7. Photograph of 48-hour growth from leg of &^ay chick embryo. Os. vap , iron hem., and eosin. 4 oc. 4 mm. len.s.
8. Photograph of 48-hour growth from subcutaneous tissue of 10-day chick embryo. Os. vap., iron hem. 6oc. 4 mm. lens.
9. Photograph of 48-hour growth from heart of 7-day chick embryo. Os. vap., iron hem. 4 oc. oil-imm. lens.
1. Camera-lucida drawing of cell, showing primitive fibrils in the cytoplasm. 48-hour growth from subcutaneous tissue of 9-day chick embryo. Os. vap., iron hem., and eosin. 4 oc. oil-imm. lens. Drawn by Miss J. E. Lovett.
2. Camera-lucida drawing of cells and fibrils united into bundles. 72-hour growth from subcutaneous tissue of 11 -da}' chick embryo. Os. vap., iron hem., and eosin. 4 oc. oil-imm. lens. Drawn by Miss J. E. Lovett.
3. C^amcra-lucida drawing showing cells with fibrils within the cytoplasm and also fibers. 120-hour growth from subcutaneoas tissue of 11-day chick embryo. Os. vaj) , iron licm., and eosin. 4 oc. oil-imm. leiiM. Drawn by Miss J. E. Lovett.
4. Camera-lucida drawing of cells from 24-hour growth of leg of 8-day chick embryo. Os. vap., iron hem., and eosin. 4 oc. oil-imm. lens.
5. Camera-lucida drawing of cell undergoing mitosis in 48-hour growth from subcutaneous tissue of 11-day chick embryo. Os. vap., iron hem., and eosin. 4 oc. oil-imm. lens.
6. Camera-lucida drawing of smooth muscle-cells, showing myofibrils from 4S-hour growth of amnion of .")-day chick embryo. Os. vap., iron hem. 4 oc. oil-imm. lens.
7. Camera-lucida drawing of cell from 24-hour growth of 6-day chick embryo, showing deposit along surface of the cell, which is stained like mitochondria with Bensley's aniline fuchsin, methylene green stain. 4 oc. oil-imm. lens.
8. Camera-lucida drawing of tendon-cclLs from 72-hour growth of muscle of 9-day chick embryo. Zenker's fixation — Mallory's stain. 4 oc. oil-imm. Icils.
9. Camera-lucida drawing of epithelial cells of 48-hour growth froin amnion of 5-day chick embryo. Iodine-vapor fixation — Mallory's stain. 4 oc. oil-imm. lens. 10 Camera-lucida drawing of thin membrane of connective tissue from 72-liour growth of 10-day cliiek embryo. Silver nitrate and Ehrlich hemato.xylin. 4 oc. and oil-imm. lens.