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The connective tissue framework of the femur in mice of different ages.

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The Connective Tissue Framework of the Femur
in Mice of Different Ages
Medical Research Center, Brookhaven Naticmal Laboratory,
Upton, Long Island, New York
Employing the methods of electron
microscopy, Robinson ('60) , Robinson and
Watson ('55), Fitton Jackson ('60), Martin ('54), and other investigators have
presented a concise submicroscopic picture
of bone and cartilage matrices. These
studies have been made on ante partum
as well as post partum laboratory animals.
Young cartilage is characterized by a delicate network of fibers. Cartilage of older
animals is conspicuous even with light
microscopy and is seen organized into
fiber bundles and systems. In as much as
it would be difficult to trace the deployment of fiber bundles by the electron
microscope, histochemical staining methods and polarized light have been used in
this investigation, not to analyze the birefringence of these structures but to enhance contrast for observation. The present
study was initiated, therefore, to determine
the presence of the fibrous framework at
the distal end of the femur in mice of different ages.
Forty-five female mice of the Brookhaven National Laboratory strain of Swiss
Albino were sacrificed in groups of nine
at 1, 5, 8, 26, and 52 weeks of age. Both
femora together with the attached proximal third of the tibia were removed from
each animal. The tissues were fixed for
three hours in a 3: 1 mixture of absolute
ethyl alcohol and glacial acetic acid. Extended fixation was carried out for 24
hours in a ten percent formol-saline solution. Fixed samples were then washed for
24 hours in running tap water (Pelc and
Glucksmann, '55). Decalcification was
carried out in a ten per cent solution of
disodium versenate (Birge and Imhoff,
'52). Paraffin blocks were finally prepared
ANAT. REC.,149: 559-576.
and 5 p longitudinal sections were cut.
The following histochemical procedures
and staining methods were employed:
Periodic acid-Schiff method (Hotchkiss,
'48) for glycogen and neutral mucopolysaccharides, Rinehart and Abul-Haj's ('51)
colloidal iron method for acid mucopolysaccharides, toluidine blue 0 method (Ham
and Harris, '50) for metachromasia, Foot's
modification of Bielschowsky's stain (Mallory, '42) and hematoxylin and eosin. A
set of sections were treated with diastase
(Gurr, '59) prior to staining with periodic
acid-Schiff in order to eliminate the presence of any glycogen. Additional sets of
sections were treated with testicular hyaluronidase (Greulich and Friberg, '57)
prior to staining with toluidine blue 0,
colloidal iron and periodic acid-SchS. All
the preparations were studied with polarized light as well as with the light microscope.
Histochemical staining
The y metachromatic reaction which
appeared following toluidine blue staining
of the distal epiphyses was abundant at
the epiphyseal disk, the cartilaginous core
of metaphyseal trabeculae and the articulating surface of the epiphysis. In young
animals the reaction was abundant in cartilage matrix. Older animals revealed nonmetachromatic fiber systems within the
matrix. The fiber systems were most prominent at the metaphyseal trabeculae, articular surface and epiphyseal disk and were
recognized more by their lack of staining
(figs. 3 , 4 ) .
lRe?e?rch supported by the U. S. Atomic Energy
Presented in part at the 1st International Congress
of Histochemistry and Cytochemistry. in Pans, August
31, 1960.
Colloidal iron reaction parallels the toluidine blue staining in that both reactions
diminished in intensity with increasing
age. The colloidal iron reaction stained
sulfated mucopolysaccharides blue and
connective tissue collagen red. With increasing age the collagenous fiber systems
present within cartilage matrix were
clearly observed and traced (fig. 5 ) . At
the metaphysis intense collagen staining
was seen about active osteoblasts lining
the trabeculae (fig. 7). In addition to the
osteoblastic collagen which surrounds active osteoblasts, clearly demonstrable collagen fiber systems were observed within
the cartilaginous trabeculae. These fiber
systems were observed throughout the entire length of the trabeculae (fig. 7 ) and
differed in orientation from the thick
masses of collagenous elements seen about
active osteoblasts and in cortical bone
(fig. 6). Periodic acid-Schiff staining before and after diastase treatment was of
little value in observing the fibrous nature
of bone and cartilage matrix.
Bielschowsky’s silver stain more clearly
demonstrated the orientation and disposition of fiber systems which occurred in
bundles. These fiber systems were observed in the epiphyseal and articular cartilage. They appeared within and parallel
with the metaphyseal trabeculae. Trabecular fiber systems were arranged into
thick compact bundles at the base of the
epiphyseal disk. As the fiber bundles
crossed the epiphyseal disk they spread out
so extensively between columns of cartilage cells that their course became less
clearly demonstrable (fig. 8). In young
animals the fibrous nature was not marked.
In successively older animals, the fibrous
pattern became more obvious, however, the
course of such fiber systems was cut short
by the appearance of bone from the secondary center of Ossification. At the periphery one was able to follow the fiber
systems to within one or two layers of cells
of the articulating surface of the epiphysis.
At the articulating surface, surrounding
the secondary ossification center, the same
delicate fibrous pattern appeared which
was observed at the epiphyseal disk.
Polarized light studies
The microscopic examination of silver
stained sections of cartilage matrix with
partially polarized light using a first order
red retardation plate, revealed most clearly
the direction of the fibrous elements of the
femur. Non-anastamosing collagenous elements within the cartilage matrix are
shown in figure 9. These fiber systems
ran a course parallel with the metaphyseal
trabeculae, and perpendicular to the epiphyseal disk and articulating surface of the
epiphysis. One or two cell layers from the
free edge of the articulating surface, the
fiber systems appeared to arch so that the
fibers reach the joint surface obliquely
(fig. 1 3 ) . These fiber systems could not
be followed to the joint surface by the
histochemical methods used (fig. 4).
Polarized light study of unstained tissue
sections exhibited poorly birefringent trabeculae, epiphyseal disks and articulating
surfaces. Decalcified regions were also
weakly polarizing. Red blood cells present
at the subepiphyseal disk region were
strongly birefringent. Colloidal iron or silver staining was ineffective in intensifying
the birefringence. Toluidine blue stained
sections, on the other hand, enhanced very
strongly polarization of light in many of
the structures which polarized light poorly
or not at all. With the addition of a first
order red retardation plate a variety of
colors and color combinations were produced. A number of tissue components
were identified on the basis of these colors.
This method has the advantage of exhibiting in different colors, similar fibrous elements which run perpendicularly to one
another, thus making it possible to trace
the deployment and organization of the
fibrous system. With the proper orientation
of the retardation plate one half of the
cartilage matrix of the epiphyseal disk
appeared a bright apple-green color, while
the other half was cream colored (fig. 10).
Diaphyseal and epiphyseal bone was seen
in blue and yellow. The color depended
upon the orientation of the collagenous
elements (fig. 10) and the retardation
plate. Red blood cells exhibited a typical
brightly colored Maltese cross (fig. l l ) ,
while the background was a reddish-purple.
In young mice the birefringent elements
of the fibrous periosteum at the meta-
56 1
physeal region were observed traversing maintained that the orderly arrangement
the osteogenic layer, running up the ossi- is a primary requirement for metachrofying trabeculae, entering the epiphysis be- masia. Polarization of light is therefore
tween columns of cartilage cells and ter- intensified.
minating at the articulating surfaces (figs.
Through the techniques of electron mi11-14). This does not mean, however, croscopy, Robinson ('60), Robinson and
that these fiber bundles are continuous. It Watson ('55), Fitton Jackson ('SO), Martin
was noted that in longitudinal sections the ('54), and other investigators have furfiber systems appeared to originate within nished an interesting submicroscopic view
cartilage matrices. The fibers, most of of cartilage and of bone matrix. Young
which ran longitudinally, appeared to cross cartilage matrix is generally made up of a
each other and fan out across the epiphy- fine meshwork of unbanded fibers. But
sis. Because of the orderly arrangement of within a short period of time, fibers exhibfiber systems, a neutral axis can be drawn iting the typical collagen spacing of 640"
extending down the center of the shaft and A appear. As the animal becomes older
curving beyond the epiphyseal disk to the collagen fibers become more conspicuous
posterior aspect of the epiphysis (see fig. and become preferentially oriented into
2). The fiber systems which are found at bundles (fig. 8). In the immediate vicinity
the anterior aspect of the neutral axis of active osteoblasts Robinson ('60) has
serve as the vertical fibers of this part of shown the accumulation of thick collagethe disk and when the axis is crossed, nous fibers (fig. 6 ) . Both types of fibers
these same fiber systems serve in part as arranged in fiber systems can be shown
the horizontal supports. The fiber systems in figure 7.
found at the posterior aspect serve as the
Ranvier (1873, 1875) as far back as
vertical fibers of this part of the disk and 1873, noted the appearance of curved fiber
when the neutral axis is crossed, serve as systems bridging the bony diaphysis and
the horizontal fibers.
the epiphyseal cartilage. Such systems
In older animals, as the fibrous ele- were concave towards the epiphysis. In
ments became easily demonstrable with 1878 Schafer (1878) described linear fiber
the low powers of the light microscope, systems passing from the epiphysis to diathe degree of polarization became in- physeal bone, and questioned Ranvier's
creased. The interwoven nature of the findings. It is entirely possible that Ranfiber systems is seen in figures 12 and 13. vier's observations pertain to the fiber sysAt the base of the epiphyseal disk, dis- tems of attachment of the patellar tendon
integration of hypertrophied chondrocytes and medial ligaments of the tibia described
was accompanied by the breaking of fiber by LaCroix ('49) in the rabbit and Pratt
systems, thus opening up the lacunae to ('59) in the rat. Weidenreich ('30) dethe medullary canal. The cartilage tra- scribed fibers continuous with those debeculae contained conspicuous longitudinal scribed by Ranvier, running longitudinally
fiber systems.
within the matrix of perichondrial bone of
fetal femora. However, Pratt ('59) disDISCUSSION
agreed with this description and stated
The fibrous nature of the femur was that these fibers were continuous with the
demonstrated at different ages by the vari- limiting network of the floor of the ossificaous staining techniques used, however, the tion groove and thus were not directly conarrangement of these fiber systems was tinuous with the fiber systems described by
observed by polarized light following tolui- Ranvier. Pratt ('59) further described the
dine blue staining. Since cartilage matrix presence of periochondrial connective tiscontains sulphated acid mucopolysaccha- sue fiber bundles which became incorporides and therefore exhibits y metachro- rated into the epiphyseal cartilage. These
masia, a high degree of dye polymerization fiber bundles were seen in the distal femur
takes place (SylvCn, '54). The association of the 23 day fetal rat and are a continuaof the substrate with the dye results in an tion of the fibrous periosteum.
orderly aggregation of molecules (MichA number of technical difficulties exist
aelis and Granick, '45). Sheppard ('42) in studying fiber systems which in our
contention have contributed to the differences in opinions and observations recorded in the literature. The stainability
of connective tissue fiber systems varies
with the disposition of the fibers in soft
tissue, mineralized tissue or cartilage. The
degree of compactness or looseness adds
difficulty in observing continuity of fiber
systems. Another difficulty exists in following the development and penetration of
fiber systems from one age to another because of their rapid growth, and still another difficulty lies in the limited resolution
of the light microscope. It was shown by
Martin ('54) that during embryonic and
post-embryonic development, cartilage matrix undergoes structural changes in its
fibrous components which are visible only
by the electron microscope.
We have seen in the present study, the
presence of a fibrous meshwork extending
from the fibrous periosteum at the perichondrial region to within a few cells of the
articulating surfaces of the epiphysis. It is
not necessary to assume, however, that the
appearance of the bicolored interwoven fiber pattern seen with polarized light (e.g.,
fig. 13), is produced by two independent fiber systems. A single fiber bundle running
in different directions between cells produces the same effect. With increasing age
the fibrous elements were more abundant
and more easily recognized microscopically.
There appeared to be at least two types of
collagenous fiber systems. Those which
are associated with osteoblasts and ossification found along ossifying trabeculae
and shaft bone, and additional fiber systems which were in association with cartilage cells and the fibrous periosteum, but
not necessarily derived from the latter.
Osteogenic collagenous fiber systems obscured the orientation and presence of
fiber systems in regions where both appeared.
It seems possible that vertically and
horizontally arranged fiber systems are
concerned primarily with orientation and
support. Figures 8, 15 and 16 show the
orientation of epiphyseal disk cells and
ceIls of the articulating surface of the distal femoral epiphysis. It was noted that
the columnar appearance of epiphyseal
disk cells was flanked by the presence of
numerous fiber bundles. Dividing carti-
lage cells, therefore, can be preferentially
oriented into columns. This organization
seems to allow the least resistance. During
growth new fibers must be added, as well
as cells, and in an organized manner. It
seems possible that existing fibers would
participate in the polymerization and organization of newly deposited cartilage
matrix. Therefore, cells are supplied for
longitudinal growth in a linear arrangement maintained by the presence of the fiber systems. If this statement is true one
may raise the question, why is it that before these fiber systems are seen with the
light microscope, cells of the epiphyseal
disk are already well columnated? Evidently oriented fibers are already present
as has been shown with the electron microscope and polarized light studies. It appears that the shape of the epiphyseal disk
can also be maintained by the fibrous organization (figs. 15 and IS), in addition
to the organization of cartilage cells which
are arranged radially at the articulating
surface (fig. 16). This particular arrangement of cells allows for a radial expansion
Fig. 1 The diagram represents a pattern of
stress lines determined mathematically and resulting from loading the head of a human femur.
Note the similarity between the organization of
the stress lines, the trabecular arrangement in
figure 17 and the fibrous pattern in figure 2.
(Reproduced by permission from John C. Koch,
Am. J. Anat., 21: 177-298, '17.)
of the condyles during growth. As the
cartilage cells of the epiphyseal disk hypertrophy and finally degenerate, cartilage
spicules which will eventually become ossified are left behind supported by bundles
of fibers (fig. 7). This support is also extended to the cartilaginous disk and articulating surface of the epiphysis.
Koch in 1917 determined mathematically the lines of stress in human femora
resulting from loading. He asserted that
the calculated stress lines accounted for
the actual arrangement of the bony trabeculae (figs. 1 and 17). It may well be that
stress lines are important in regulating the
direction and final alignment of these fiber
systems, not only at the trabeculae but at
the epiphysis as well. It has been shown
in the present study that the lines of stress
which have been demonstrated by Koch
appear to be similar to the general arrangement of the fiber systems demonstrated in
figure 2. However, weight bearing is not
essential, at least initially, in producing the
stress necessary for the organization of the
fiber system, since we have observed this
fibrous system even in the bones of very
young human fetuses. On this basis, it
seems plausible to believe that the fiber systems can play an important role in the s u p
port of the epiphyseal structures, in addition to maintaining the proper orientation
of cells during growth. As the animal becomes older and the individual fibrous element becomes more mature, increased support is afforded for greater weight bearing.
Histochemical staining methods and
polarized light were used to study the organ-
Fig. 2 A diagram representing the arrangement of the supporting fibrous system at the
distal end of the femur. A neutral axis can be drawn along the length of the femur. Note
especially that the fiber systems anterior to the neutral axis cross the epiphyseal disk vertically and become horizontal fiber systems of the disk when on the opposite side of the
neutral axis. The same is true of the posterior fiber systems which run vertically across
the disk then horizontally when the neutral axis is crossed.
ization of the fibrous framework of the
distal femoral epiphysis in the mouse.
Fiber systems were shown within the metaphyseal trabeculae and epiphysis. These
fiber systems were arranged in an organized fashion. The orientation of the metaphyseal trabeculae, cells of the epiphyseal
disk and articulating surface may well be
attributed to the deployment of these fiber
systems. If so, the arrangement of fiber
systems at the disk will allow for a longitudinal type of growth, while the arrangement at the articulating surfaces will allow
for spherical expansion of the condyles.
In view of these experiments, fiber systems
appear to play an important role in the
architectural support of the epiphyseal
The author wishes to express his gratitude to Dr. E. P. Cronkite for his advice
and constructive criticism, to Miss M.
Pavelec for her technical assistance and
Mr. R. E. Smith for the photography.
Birge, E. A., and C. E. Imhoff 1952 Versenate
as a decalcifying agent for bone. Am. J. Clin.
Pathol., 22: 192-193.
Fitton Jackson, S. 1960 Fibrogenesis and the
Formation of Matrix: In: Bone as a Tissue. K.
Rodahl et al., ed. McGraw-Hill, New York, pp.
Greulich, R. C . , and U. Friberg 1957 Histochemical studies of sulfomucopolysaccharides
in the organic matrices of meneralized tissues.
Exp. Cell Res., 12: 685-689.
GUR, E. 1959 Methods of Analytical Histology
and Histo-Chemistry. Williams and Wilkins Co.,
Baltimore, p. 52.
Ham, A. W., and W. R. Harris 1950 Histological
technique for the study of bone and some
notes on the staining of cartilage In: Jones R.
McClung, McClung's Handbook of Microscopical Technique. P. B. Hoeber, Inc., New York,
pp. 269-284.
Hotchkiss, R. D. 1948 A microchemical reaction resulting in the staining of polysaccharide
structures in fixed tissue preparations. Arch.
Biochem., 16: 131-141.
Koch, J. C. 1917 The laws of bone architecture.
Am. J. Anat., 21: 177-298.
Lacroix, P. 1949 Origine, structure et Valeur de
1'Encoche d'ossification de Ranvier. Bull. Histol. Tech. micr., 18: 156-169.
Mallory, F. B. 1942 Pathological Technique,
W. B. Saunders Co., Philadelphia, p. 161.
Martin, A. V. M. 1954 A n electron microscope
study of the cartilaginous matrix in the developing tibia of the fowl. J. Embryo1 Exp. Morph.,
2: 38-48.
Michaelis, L., and S. Granick 1945 Metachromasy of basic dye stuffs. Am. J. Chem. SOC.,
67: 1212-1219.
Pelc, S. R., and A. Gliicksmann 1955 Sulphate
metabolism in the cartilage of the trachea,
pinna, xyphoid process of the adult mouse as
indicated by autoradiographs. Exp. Cell Res.,
8: 336-344.
Pratt, C. W. M. 1959 The significance of the
perichondrial zone in a developing long bone
of the rat. J. Anat., 93: 110-122.
Ranvier, L. 1873 Quelques Faits ReIatifs a u
DBveloppment du Tissu Osseux. Compt. rend.
Acad. Sci. Paris, 77: 1105-1109.
1875 Trait6 Technique d'Histologic.
Paris, F. Savey.
Rinehart, J. F., and S. K. Abu'l-Haj 1951 An
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of acid mucopoIysaccharides in tissues. Arch.
Path., 52: 189-194.
Robinson, R. A. 1960 Chemical Analysis and
Electron Microscopy of Bone, In: Bone as a Tissue. K. Radahl et al., editors. McGraw-Hill,
New York, pp. 186-250.
Robinson, R. A., and M. L. Watson 1955 Crystalcollagen relationships in bone a s observed in
the electron microscope. I11 Crystal and collagen morphology as a function of age. Ann.
N. Y. Acad. Sci., 60: 596-628.
Schafer, E. A. 1878 Notes on the structure and
development of osseous tissue. Quart. J. micr.
Sci., 18: 132-141.
Sheppard, S. E. 1942 The effects of environment and aggregation on the absorption spectra
of dyes. Rev. mod. Phys., 14: 303-340.
SylvBn, B. 1954 Metachromatic Dye-Substrate
Interactions. Quart. J. micr. Sci., 95: 327-358.
Weidenreich, F. 1930 Das Knochengewebe. In:
Handbuch der Mikroskopischen Anatomie des
Menschen. W. von Mollendorff editor, Julius
Springer Pub. Berlin. Band 2, Teil 2.
3 Toluidine blue staining of metaphyseal trabeculae of the distal
femoral epiphysis of a n eight-weeks-old mouse. Note the fibrous
nature of the trabeculae. These fiber bundles run parallel with the
trabeculae. The clear white areas represent decalcified regions.
X 400 oil immersion.
The fibrous nature of the articular surface of the distal femoral epiphysis of a 52-weeks-old mouse is revealed after toluidine blue staining. X 240 oil immersion.
A distal femoral epiphyseal disk of a 52-weeks-old mouse stained with
colloidal iron. Note that at this age the disk is essentially negative,
however, the fibrous structure is prominent. x 40.
A decalcified femoral cortical bone of an eight-weeks-old mouse is
shown in this photomicrograph. Collagenous fibers are being deposited
about active osteoblasts. The fiber bundles mark the limit of calcification and are negative for colloidal iron staining. They stain red
typical of collagen. x 240 oil immersion.
Edgar A. Tonna
7 The photomicrograph demonstrates the delicate supporting fiber systems found running parallel and within the ossifying metaphyseal
trabeculae of the distal femur of an eight-weeks-old mouse. Colloidal
iron staining. x 240 oil immersion.
8 Collagenous fiber systems are seen in the cartilaginous matrix from
metaphyseal trabeculae of an eight-weeks-old mouse. Stained with
Foot’s modification of Bielschowsky’s method and photographed under
partially polarized light. X 240 oil immersion.
By tearing of cartilage matrix, individual non-anastamosing fiber
systems can be demonstrated. Same specimen and similar preparation as figure 6. X 600 (oil immersion) initial magnification enlarged to X 1,800.
Edgar A. Tonna
A n eight-weeks-old distal femur of mouse photographed under polarized light, after toluidine blue staining. Notice that the matrix of the
epiphyseal disk appears apple-green a t the left half of the photomicrograph and cream colored at the right half. Decalcified bone appears
blue or yellow depending upon the orientation of the collagen. x 40.
A higher magnification of the trabeculae and epiphyseal disk taken
from the distal femur of a n eight-weeks-old mouse. Fiber bundles can
be shown in two colors, namely, apple-green and cream. Note that
the colored bundles running across the disk appear to be broken in
numerous places. The spaces represent the different arrangement of
fibers not exactly parallel to the other direction, and, therefore, cannot be seen with the slide i n this orientation. Toluidine blue stained
and viewed under polarized light. x 160 oil immersion.
12 The photomicrograph of a n eight-weeks-old distal epiphysis reveals
under polarized light the interlaced nature of the fibrous structure.
Toluidine blue staining. X 40.
The interlacing fibrous structure terminating at the articulating surface of the distal femoral epiphysis in a n eight-weeks-old mouse is
shown under polarized light i n different colors. Toluidine blue staining. x 160 oil immersion.
Edgar A. Tonna
Although the fibrous structure of mouse epiphysis cannot be readily
seen with the usual staining procedures a t birth, toluidine blue staining and polarized light reveal a well formed pattern shown by the
arrows. x 52.
15 The photomicrograph shows a toluidine blue stained distal femoral
epiphyseal disk of a n eight-weeks-old mouse. Notice the direction of
the arrows which are parallel to the penetrating fiber systems. The
particular arrangement of the fiber systems appears t o allow for the
linear orientation of dividing cartilage cells. Compare with figures 1,
2, and 17. X 52.
Edgar A. Tonna
16 A different region of the same sample shown in figure 15. Note the
direction of the arrows especially at the articulating surface. The
direction of the fiber systems here account for the spherical expansion
of the distal femoral epiphyseal condyles. Compare with figures 1, 2
and 17. x 55.
5 74
The arrangement of the bony trabeculae is seen i n a section of the
proximal end of a human femur. Note that the trabeculae correspond with the stress line pattern in figure 1 and bear strong resemblance to the fibrous pattern shown in figure 2. (Reproduced by permission from John C. Koch, Am. J. Anat., 2 1 : 177-298, 1917.)
Edgar A. Tonna
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