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With 6 Text figure ansd 7 Tables. | With 6 Text figure ansd 7 Tables. | ||
The American Journ. of Anat. Vol. 5. 433 - (1906) | |||
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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,<ref>Rambaud & Renault, Origin et Devel. d. Os., Paris, 1864.</ref> but the small dried arms pictured in Bell’s Anatomy<ref>Bell’s Anatomy, New York, 1834, p. 66.</ref>and in Rambaud & Renault’s Atlas<ref>Rambaud & Renault, Origin et Devel. d. Os., Paris, 1864.</ref> 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,<ref name="Schultze1897">Schultze, Grundriss d. Entwickl. d. Menschens, 1897.</ref> from transparent embryos<ref name="Schultze1897">Schultze, Grundriss d. Entwickl. d. Menschens, 1897.</ref> or from reconstructions<ref>Gaupp, Hertwig’s Handbuch d. Entwickelungslehre, Jena, 1905; Bardeen, Amer. Journal of Anatomy, IV, 1905.</ref> are taking their place. | 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,<ref>Rambaud & Renault, Origin et Devel. d. Os., Paris, 1864.</ref> but the small dried arms pictured in Bell’s Anatomy<ref>Bell’s Anatomy, New York, 1834, p. 66.</ref>and in Rambaud & Renault’s Atlas<ref>Rambaud & Renault, Origin et Devel. d. Os., Paris, 1864.</ref> 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,<ref name="Schultze1897">Schultze, Grundriss d. Entwickl. d. Menschens, 1897.</ref> from transparent embryos<ref name="Schultze1897">Schultze, Grundriss d. Entwickl. d. Menschens, 1897.</ref> or from reconstructions<ref>Gaupp, Hertwig’s Handbuch d. Entwickelungslehre, Jena, 1905; Bardeen, Amer. Journal of Anatomy, IV, 1905.</ref> 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<ref>Hill,JohnsHopkins Hospital Bulletin, 1906.</ref> 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.<ref>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.</ref> 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 [[Histology_Stains#Mallory_Trichrome_Stain|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. | |||
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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)
| Historic Disclaimer - information about historic embryology pages |
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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.
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.
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 3) 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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