Paper - On Ossification Centers in Human Embryos
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=On Ossification Centers In Human Embryos Less Than One Hundred Days Old=
(Fromthe Anatomical Laboratory Of The Johns Hopkins University.)
With 6 Text figure ansd 7 Tables.
The American Journ. of Anat. Vol. 5. 433 - (1906)
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As the study of the bones preceded that of the other structure of the human body, so their ossification was the first subject investigated in embryology. The early anatomists became interested in the development of bones on account of the difference between them in adults and children, and it was but a step to their study, first in the fetus and then in the embryo.
More than one hundred years ago the early ossifications were studied with vigor and in a short time the subject was closed, and we may say that our present knowledge dates mostly from before 1820. With the improve ment in embryological methods so many new fields were opened that it did not seem worth while to destroy good specimens nor to make laborious reconstructions to study a subject which seemed so unpromising in re sults. However, it is apparent that there is considerable difference of opinion regarding the time of ossification as well as the number of centers in certain bones, which frequently diminish as they are studied more carefully .
We notice in looking over the older literature that the ossification was studied by means of ordinary dissection after which the very small specimens were dried upon a glass slide. Such specimens show sharply marked bone centers, and are very useful, but unfortunately the embryos are pretty well destroyed in their preparation. Furthermore, very small centers and delicate attachments are difficult to see, and this defect in the specimens has led to numerous erroneous conclusions. The time of ossification and the order of the appearance of the centers has never been definitely settled, mainly because the specimens were not numerous and were much injured in their study, and because the various investigators did not determine correctly the age of the embryos. Thus, for instance, we read in Béclard’s article that the centers for the mandible, maxilla, clavicle, humerus, ulna, radius, femur and tibia are present in an embryo 35 days old (16 lines long) which, according to my table, must be 54 days old. Numerous other embryos are studied in this article, each time giving their age in days or weeks, and usually omitting their length. A similar criticism may be made regarding Meckel’s great paper from which is derived our main information regarding the development of the spinal column and skull.[1]
The older illustrations of primary bone centers are not especially good, for in them the finer details are obscure and the enlarging glass did not aid to make them sharper. However, some of Meckel’s pictures are still used in the anatomies,[2] but the small dried arms pictured in Bell’s Anatomy[3]and in Rambaud & Renault’s Atlas[4] have not been copied extensively. Semidiagrammatic illustrations, i. e., older bones with the earlier centers marked in them have gradually taken their place. And it is only in very recent years that suitable illustrations from X-ray pictures,[5] from transparent embryos[5] or from reconstructions[6] are taking their place.
The newer methods enable us to recognize and to follow the early ossi- fication centers with much greater precision than was possible in Meckel’s time. Instead of a dissected and dried specimen we now have sections and reconstructions, and what is still better transparent specimens made according to the method of Schultze, which enable us to detect the minutest bone (0.1 mm. in diameter) and to study it in relation to the rest of the skeleton without destroying the embryo. And last, but not least, we have a standard of measurement (the crown-rump line of His) from which we can determine the age of the embryo with an error of but a few days. It naturally follows that all that is now required is a good collection of transparent embryos to determine the time of appear- ance and order of development of the bone centers.
During the past half dozen years I have cleared from time to time human embryos which were shrivelled or otherwise made unfit to cut into serial sections. Gradually the number increased so that now I have some 60 transparent specimens in my collection with crown-rump meas- urements ranging from 10 to 110 mm. in length. Most of these sneci- mens are used in this study.
We have gradually learned that it is best to clear specimens which have been well shrivelled in alcohol in a 1% solution of KOH for a few hours and not in the strong solution recommended by Schultze. With the weaker solution the tissues, of the smaller embryos especially, remain firm, and, in the end, the specimen is perfectly transparent with all the bones held in place. After the treatment with potash the embryo is placed in the following solution for days, or even for months:
- Water - 79
- Glycerine - 20
- Potash - 1
From time to time the embryo may be returned to a 3% solution of potash for a number of hours in order to hasten the process. T'he action of the glycerine has been to make the tissue more resistant, and for this reason the strength of the potash is increased. In case there is much blood pigment in the embryo or it is otherwise colored through age, this may be removed by placing the specimen for a number of days in the strongest ammonia to which a little potash has been added, as recom- mended by Hill[7] In case the embryo is stained with alum cochineal before it is cleared the bones alone are colored red, and for the study of their finer connections this is often an advantage.
Many of the embryos received recently have been preserved in formalin and for a loqg time it was practically impossible to clear them in the ordinary way. Finally, in desperation, I placed such an embryo in a 10% solution of potash and to my astonishment I found that it began to clear at the end of a month. By further treatment it was found that such embryos could be cleared perfectly well, and in case the bones are not decal- cified by the formalin the very best specimens are obtained. In them the tendons and other white fibrous tissue are rendered tougher than in the specimens treated with alcohol alone. Finally when the embryos are well cleared they are gradually transferred to stronger and stronger glycerine until all the water is removed from them.
Before clearing the embryo it is well to cut it through the sagittal plane into two equal parts, for by this treatment the bones are all brought into view in the finished specimen. Later these halves may be fixed to glass slides with gelatin, as recommended by Bardeen. The specimen is to be taken from pure glycerine and wiped gently and then placed upon a glass plate which is covered with melted gelatin. As soon as the gelatin is cool this slide with the embryo attached is returned to pure glycerine which extracts the water from the gelatin and makes it very firm. The ossification centers.show best when viewed with a large lense over a dark background in direct sunlight.
I have made numerous attempts to study the ossification centers in serial sections stained in carmin and in hematoxylin, but find that they are by no means as satisfactory for this purpose as are the Schultze specimens. An embryo 20 mm. long (No. 22), which we have studied with great care shows.no ossification centers in the sections, while embryos 15 mm. long show them when cleared.[8] The model made by Ziegler, which is from Hertwig’s reconstruction of an embryo 80 mm. long does not show the bones of the skull developed to as great an extent as they are in cleared specimens. So for the present the Schultze method en- ables us to locate the first bones with much greater certainty than do sections, with the possible exception of those colored with Mallory’s connective-tissue stain.
Ossification Centers in the Second Month
All anatomists agree that the clavicle is the first bone to ossify and that it appears about the middle of the second month or during the sixth week. Béclard states that he found it in an embryo 30 days old, but as E. H. Weber remarks in Hildebrandt’s Anatomy, he has probably under- estimated the age of the embryo. Judging by the number of ossification centers present Béclard’s embryo must have been 44 days old, for it corresponds with my specimens 263, C and A. Meckel must also have underestimated the age of an embryo in which the clavicle was found to be three lines long, for he places it in the middle of the second month. Ac- cording to my reckoning, his specimen is fully 56 days old, for it corresponds with my specimen 263, b. 2, Table 11. Similar differences of opinion will be found regarding the time of ossification of all sharply defined bones due, no doubt, to the difficulty in determining the age of embryos 100 years ago. Therefore, little is to be gained in reviewing the literature upon this subject for such specimens, since the results are not satisfactory when compared with a good series of Schultze specimens. Especially is this true regarding the literature of obscure bones which are said to arise from more than one center. In my description I shall, therefore, confine myself closely to my own specimens.
Table I shows at a glance the ossification centers which appear during the second month. Five embryos of the 39th day were cleared, and in two of them the clavicle and mandible are found, while in one it is uncertain whether or not the clavicle is ossified. When the question mark (?) is inserted in the table, for instance for specimen E, it indicates that a hyaline body is seen, but that it is not as white as an ossification center should be. During the 40th day the maxilla also appears, to which are added the humerus and femur on the 42d day. Next come the radius and tibia on the 44th day and then the ulna on the 49th day. Several days elapse before the fibula appears, as was known to the older anatomists. In less than a day later the scapula follows and on the 56th day the supraoccipital begins to ossify. During the 55th day the ribs make their appearance, and judging by their size, it is the 6th and 7th which ossify first. The ossification spreads rapidly in both directions and on the next day (56th) it is present in all of the ribs, with the exception of the 1standthe12th. On the 56th day we also find the beginning of ossification in the frontal, zygomatic, squamo-zygoniatic and parietal bones in the skull, thc ilium in the lower extremity and the terminal phalanx of the thumb in the hand.
Fig.1. Embryo No. 333, enlarged 5 times. The outlines of the embryo were taken from a fresh specimen.
The remarkable regularity of the appearance of the bones make of them the best index of the size and age of an embryo we now possess. The measurement of the crown-rump length of an embrvo is modified by the method of preservation and the amount of distortion, but neither of these factors influences the order of ossification. I may add that the length of the embryo was determined by direct measurement from crown to rump, but in case the embryo was distorted this measurement was de- duced from the neck-rump length which is the least variable of external measurements. The age of the embryos in days was obtained by multiplying the square root of the crown-rump length in millimeters by ten.[9] These ages correspond with the estimations given by embryologists. If in the course of time our data enable us to determine the ages more accurately, the ages I have ascribed to the various embryos can easily be changed, for in all the specimens the length from crown to rump is also given.
Ossification Centers in the Skull
Mandibula
The mandible appears a very little later than the clavicle. In two embryos, 15 mm. long, 39 days old, it is present as a finely granular or reticular mass, half a millimeter long, immediately below the epidermis towards the free end of the first arch, representing, of course, the body of the jaw. In the next specimen, 263, b, the mandibula is found to be a slender but compact bone about one millimeter long reaching nearly to the midventral line; in D it is a little larger and more sharply defined. On the 42d day (No. 42) the bone measures 2 mm. and shows the beginning of the ramus and of the alveolar process. It now gradually enlarges, measuring 3 mm. in No. 56 and No. 333. The lower jaw in these embryos is thin and transparent, showing a delicate structure. The alveolar process and the ramus are very transparent, while the base and the part of the body near the midventral line is thick and opaque. Much the same appearance is seen in No. 202 (55 days), while in 274 the jaw is fully 5 mm. long and shows the beginning of the coronoid process and the condyle. A day later, No. 263, b, 2, the jaw is 6 mm. long, and within it can be seen three lines of thickened bone radiating from the symphysis, one towards the angle, one into the condyle, and one into the coronoid process. The next day a few sockets for the teeth may be seen in the alveolar process. This condition remains, the jaw only growing in size, until the 58th day (272) when the jaw appears hollow and the three radiating lines no longer reach to its anterior end. By the 7'5th day (288, b) the jaw has grown to be 10 mm. long and meets its fellow to form the symphysis on the midventral line. The condyle has become much more sharply defined and its bone fibers again reach to the symphysis. About this time the mylohyoid line and the lingula appear. On the 83d day the ramus is becoming relatively thinner and broader, the coronoid process has moved farther away from the con- dyle, the angle has become more marked, and the alieolar process has in- creased in length. The mandibula has now its characteristic shape, and measures 14 mm. in length. In older embryos its form is characteristic; it gradually increases in size, measuring 19 mm. in embryo P.
Maxilla
The maxilla, according to my specimens, arises from two centers only, one to form the premaxillary bone and one to form the main body. In one of the youngest specimens (2G3, b) the maxilla is marked by a mass of granules, together one-half millimeter in diameter, lying spread out just beneath the eye and one millimeter from the middle line. In another specimen (C) it is impossible to find the maxilla, but a very small premaxillary is found measuring one-fourth of a millimeter in diameter. A second specimen of the 42d day (42) shows both centers present as granular masses. The maxilla is just beneath the eye, over a millimeter in diameter and the premaxillary is as small as in C and separated by a millimeter from the maxilla. A little later (56 and 202) the two bones are denser in structure and both have parallel processes which no doubt are to form the frontal process. These two bones are found united along the alveolar border on the 56th day (274), but the frontal processes remain separated for a long time. In another embryo of the 56th day (263, b, 2 ) the frontal process is found to be distinctly double, one-half coming from each center, and the alveolar process is large and includes both of them. The orbital plate and the palatine process are just beginning and the zy,gomatic process is well developed. By grasping the different sides of the bone between two needles it is easily demonstrated that there is but one bone from this time onward. If much pressure is exerted it is observed that the bone bends, and if too much, it breaks. In later stages when the palate, temporal and zygomatic bones are present it is easily seen that these are separate, although they come in contact with the maxilla. At no time are more than two centers present, and these unite in the very beginning of the third month. In Table I1 the parallel lines are inserted between the columns for the maxilla and the premaxillary for specimens, in which these two bones can still be recognized, but they are firmly united. However, these processes, especially the zyogmatic, are easily broken off, and, judging by the illustrations of this bone in Rambaud & Renault, their numerous centers are due to such breaks. In the next specimen (266) the premaxillary is separate, and not united with the body of the maxilla; in all of the older embryos they are united.
Fig. 2. Embryo No. 284 (54 mm. long), enlarged nearly 2% diameters. Immediately beneath the squamo-zygomatic the alisphenoid may be seen.
At about this time the palate bone is well developed and overlaps the palatine process of the maxilla. A little later (263, b, 1) a small palatine process arises from the premaxillary process and in older stages (300) the bony palate reaches nearly to the vomer. In this embryo the infra-temporal and orbital plates are well formed, and the bone is well hollowed out, forming the well-marked hiatus which communicates freely with the cavity of the nose.
After the premaxillary bone joins the maxilla it is in general cubical in shape, measuring on a side 3 mm. on the 65th day; 4.5 mm. on the 75th day (288, b) ; and 6 mm. on the 85th day (300).
It is said by Rambaud & Renault that the primary ossification centers unite during the third month. Toldt states that they appear at the end of the second month and unite at the end of the fourth month. If Toldt’s statement is correct the primary centers should be present in practically all of the embryos given in Table 11. Toldt is a very reliable observer, and in order to verify his statement I tested the maxillae in all of my embryos by squeezing them between two needles, but at no time could I demonstrate more than one center, exclusive of the premaxillary. The bone was dissected out in embryo 288, b, and was found to be a single bone, and was broken with difficulty when handled with two needles in glycerine. The opinion I have reached regarding the ossification of the maxilla is fully confirmed by Ziegler’s copy of Hertwig’s model of the skull of an embryo 8 em. long, and by Schultze’s illustration of the bones of the skull of an embryo of the third month.
Occipital bone
According tothe embryos I have studied, the occipital bone arises from nine primary centers, two less than described by Meckel. On the 55th day (202)two cartilages which are thin and transparent, and may be ossified, are seen just above the foramen magnum. A little later these cartilages are fully ossified, are beginning to unite across the middle line, and lateral to them two small new centers are seen. On the next day all four centers begin to unite to form a single bone, the supraoccipital, measuring 2 mm. in height and 6 mm. in width. In the same embryo (263, b, 2) two very small exoccipitals one-quarter of a millimeter in diameter are seen. On the 57th day (266) the supraoccipital centers are well blended and the interprietals make their appearance as two reticular membrane bones, which unite duriqg the next day (263, b, 1 and 272). On the 65th day the basioccipital makes its appearance and on the 75th day all the bones above the foramen magnum are united into the single tabular part of the occipital bone. The accompanying diagram shows the general arrangement of the centers with the date of their appearance marked upon them and their time of union marked between them.
FIg. 3. Diagram of the ossification of the occipital bone. The figures upon or between the centers indicate the time in days in which they appear or unite.
Although there are over 20 perfect specimens in the cleared embryos of the proper ages, in none of them is there any sign of more than two centers for each interparietal as is asserted by Meckel, Ranke[10] and Bolk.[11] The supraoccipital is ti mm. long, crossing the middle line in an embryo 56 days old, and above it lie the interparietals which together are a little narrower and nearly as long. At this time the exoccipital is about a millimeter long. They all grow quite rapidly and on the 73d day the supraoccipital and the interparietal begin to unite. Now the former measures 4 x 10 mm. and the latter 2 x 7 mm. The common squamous portion of the occipital bone measures 6 x 10 mm. on the 75th day,12x16 on the 85th day, and 18 x 30 on the 105th day. The growth of the exoccipital and basioccipital is not so rapid, the former is 3 mm. long on the 73d day and 7 mm. on the 105th day; during the same time the basioccipital grows to measure 2 x 6 millimeters.
References
- ↑ Béclard, Meckel’s Archiv, 1820.
- ↑ Rambaud & Renault, Origin et Devel. d. Os., Paris, 1864.
- ↑ Bell’s Anatomy, New York, 1834, p. 66.
- ↑ Rambaud & Renault, Origin et Devel. d. Os., Paris, 1864.
- ↑ 5.0 5.1 Schultze, Grundriss d. Entwickl. d. Menschens, 1897.
- ↑ Gaupp, Hertwig’s Handbuch d. Entwickelungslehre, Jena, 1905; Bardeen, Amer. Journal of Anatomy, IV, 1905.
- ↑ Hill,JohnsHopkins Hospital Bulletin, 1906.
- ↑ The same is shown in Bardeen’s studies on the development of bones (Anat. Anz., XXV & Amer. Jour. Anat., IV). The earliest stage of different centers he describes are always a little later than those in which they may be seen in embryos cleared in potash.
- ↑ See Mall, Amer. Jour. of Anat., 11, 335; Johns Hopkins Hospital Bulletin, 1903; and Johns Hopkins Hospital Reports, IX, 1900.
- ↑ Ranke, Abhandl. d. K. Bayer. Akademie u. Wiss., XX
- ↑ Balk, Petrus Camper, 11
Mall, F. P. : On Ossification Centers in Human Embryos. The American Journ. of Anat. Vol. 5. 1906. http://onlinelibrary.wiley.com/doi/10.1002/aja.1000050403/abstract
Cite this page: Hill, M.A. (2024, May 4) Embryology Paper - On Ossification Centers in Human Embryos. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_On_Ossification_Centers_in_Human_Embryos
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