Eanvier recommends, as a staining fluid, purpurine, the
formula of which is as follows : Take one gramme of powdered
alum and add to it two hundred grammes of distilled water,
which boil in a porcelain dish. To this solution add some pow-
dered purpurine diluted with water. If the boiling be now con-
tinued, a portion of the purpurine will dissolve. Filter while
warm, and receive the colored fluid in a flask which contains
60 c.c. of alcohol. This liquid has a rose-orange color. The
nuclei of the corpuscles will be colored red and have a double
contour ; the cell-body will be bright red.
Hyaline cartilage may be well exhibited in the respiratory
tract of young children, as in the cricoid cartilage of an infant
two or three years old.
Yellow elastic or reticular cartilage is a very distinctive
form. It consists of the hyaline variety permeated with elas-
tic networks. Examples of it may be obtained from the human
1 See chapter on General Methods.
86
MANUAL OF HISTOLOGY.
epiglottis, laryngeal cartilages, and the pinna of the ear (Fig.
34). The presence of elastic fibres is proved by their resistance
to boiling in acids and alkalies, and their failure to color with
carmine. Sections may be made with the knife and prepared
in almost any of the ways already mentioned.
The appearances already described are not seen in the early
development of elastic tissue, but are easily identified in adult
FIG. 34. Section of the boiled and dried auricle of the human ear : a, retiform cartilage ; 6, connec-
tive tissue. (Rollett.)
life. Even then the elastic fibrils may only be found in the in-
terior of the cartilage, while at the periphery the matrix is
hyaline. Elastic cartilage is coated over with a delicate mem-
brane the pericJwndrium.
Fibrous cartilage. This variety is also known as fibril -
lated or fibro-cartilage. The matrix has probably no elastic
fibrils, but is interspersed with connective- tissue bundles. It
is found in the cartilages which make the lips of the joints, the
inter-articular cartilages, the cartilaginous deposits in tendons,
the cartilage of the symphysis pubis and of glenoid fossae, and
possibly in the intervertebral ligaments and sesamoid carti-
lages. There is often more or less hyaline material about them.
In many instances the line of distinction between cartilage and
fibrous tissue is difficult to make out. ' Where, however, dis-
tinct corpuscles can be demonstrated, the tissue may properly
be regarded as cartilage. These bodies are similar to those
seen in hyaline and reticular cartilage.
Division of the cartilage-corpuscle. A problem that has
THE CONNECTIVE SUBSTANCE GEOUP. 87
attracted the study of various histologists for a number of
years, since Leidy, in 1849, first directed attention to it, is the
mode in which cartilage-corpuscles divide. Various theories
have been afloat, each with its special supporters.
Dr. W. S. Bigelow, of this country, in 1878 reviewed the
subject carefully, pursuing his investigations on the hyaline
cartilage of the triton, tree-toad, frog, various fishes, the guinea-
pig, total pig, and the human embryo in health and disease.
His inquiries were especially concerned with reference to the
statement of Buetschli, that in the divisions of the corpuscles,
the splitting of the nucleus and cell-body are simultaneous.
As the result of Dr. Bigelow' s work, he concludes that the old
theory is still tenable, viz., that at first there is a division of
the nucleus, and that subsequently a septum is found in the
cell-body. After division takes place the matrix of the carti-
lage penetrates between the corpuscles, and thus two cavities
are formed. This view has received confirmation from very
extended and elaborate researches by Schleicher, to which
Flemming has also expressed a provisional assent.
Structure of the cartilage-corpuscle. According to Schlei-
cher the nuclei are provided with peculiar filaments and gran-
ules which undergo amoeboid movements when they are in the
act of dividing. In the cell-body of young cartilage-corpuscles
he has seen no network, such as has been described by some
later writers (Heitzmann, Klein, etc.), though in the adult tissue
peculiar linear markings are evident. He thinks that the nu-
cleus is not permeated by a network, but is homogeneous.
Reticulated appearances are apt, he thinks, to be the result of
using reagents that alter the natural quality of the tissues.
According to Flemming, the nucleus of the cartilage-corpus-
cles contains a network which gives the appearances described
as " coarsely granular."
In the drawings of this author the cell-bodies nowhere exhibit a network,
but, on the contrary, linear markings, which have often a concentric direction.
In many, the internal structure is represented as homogeneous. The conflict of
opinion now apparent in this matter, and the marked differences in the micro-
scopic drawings of the same object, make it apparent that these topics are still
to be regarded as subjudice.
Structure of the intercellular substance. According to Spina there is an intra-
cellular substance in cartilage which is directly continuous with the intercel-
lular substance, which itself exhibits an extremely delicate network. This
88 MANUAL OF HISTOLOGY.
condition, which he regards as an early form of cartilage, undergoes changes,
in so far that the intercellular network is enlarged and narrowed so as to give
the appearance of fascicles or bundles of parallel fibres. The meshes are filled
with a finely granular substance which is thought to be partly formed at the
expense of the network. The method employed in demonstrating these ap-
pearances consisted in taking the articular extremities of frog's bones, im-
mersing them three to four days in alcohol, then cutting thin sections, and
finally, examining them in alcohol.
BIBLIOGRAPHY.
TILLMANNS. Archiv f. mikrosk. Anat. X. Bd. X. p. 401. 1874.
BUDGE, A. Archiv f. mikrosk. Anat;. Bd. XIV., S. 65. 1877.
NYKAMP. Archiv f. mikrosk. Anat. Bd. XIV., S. 492. 1877.
HEITZMANN. Studien am Knochen u. Knorpel. Wien. med. Jahrb. 1872. V. and
H.'s Bericht.
BIGELOW, W. S. Arch. f. mikrosk. Anat. XVI., 2. 1878.
SCHLEICHEB. Ibid.
FLEMMING. Ibid.
KLEIN and E. NOBLE SMITH'S Atlas of Histology. 18791880.
RANVIER. Traite technique d'histologie. 1877.
BUETSCHLI. Zeitschr. f. wies. Zool. 29, p. 206.
SPINA, A. Sitzb. de k. Akad. der Wiss. Bd. LXXX., LXXXI. 1879, 1880.
CHAPTEE VII
THE CONNECTIVE SUBSTANCE GROUP. Continued.
BONE.
THEEE are two principal varieties of bone known to anato-
mists, the compact and the cancellous or spongy. The former
is found in the shafts of all the long bones of the body and
along the outer surface of all the short and flat bones. The
latter occurs in the articular extremities of all long bones and
in the interior of all short and flat bones.
Compact tissue consists of an unyielding, almost inelastic,
massive framework, which is traversed by networks of blood-
vessels and lymphatics, and perhaps by nerves. The dense
organic substance forming the groundwork of all bone ossein
is in reality nothing but a form of connective substance
almost precisely resembling ordinary fibrous tissue, but which
is evenly infiltrated with minute molecules of the carbonates
and phosphates of lime and some other inorganic salts. These
insoluble matters are so thoroughly intermixed with the fibrous
tissue that they give it great solidity, though at the same time
they restrict its flexibility, and therefore increase its suscepti-
bility to fracture.
Like other forms of the connective-tissue series, it contains
corpuscles that are disposed in a regular way between lamel-
lae, which here correspond to the fascicles of fibrous tissue. The
province of these corpuscles is doubtless the same as that of
other connective-tissue corpuscles, viz., to preside over the nu-
trition of the tissue in which they are found.
After decalcification by strong acids, such as the nitric or
muriatic, if the residue be boiled it will yield gelatin or chon-
drin.
These corpuscles that have just been described are not al-
90 MANUAL OF HISTOLOGY.
ways easily recognized, and, in fact, have often been ignored
by writers of anatomical text-books. They were not detected
for a long time, because the capsules in which they are em-
bedded received all the attention, and were even called 'bone-
corpuscles. But when it was discovered by Yirchovv that
these bodies had nuclei, and that they could be separated, to-
gether with their processes, from the bone, it was supposed
that the nutrition of the tissue was maintained through them,
acting in the capacity of hollow tubes. This view Virchow at
one time supported. Subsequently it was discovered that in-
jection fluids could be forced into the canaliculi and round
about the corpuscles, so that three facts became assured : (1)
the existence of capsules in the bony substance with radiating
and anastomosing passages, the lacunae and canaliculi ; (2) the
presence of nucleated and branched corpuscles in the lacunae ;
and of spaces (3) about the nucleated corpuscles and their
processes, suitable for the movement of fluids designed for the
nutrition of the part.
The structure of bone then became clear, and its similarity
with other connective substances well established. These bony
canaliculi extend to the wall of the Haversian canal, the great
channel conveying the blood-vessels and larger lymphatics.
Thus a lymph-canalicular system permeates the bone in close
connection with the blood-vessels, bathing every bone-cor-
puscle.
When a cross-section is made of any long bone, it will be
observed that most of the lamellae have a concentric arrange-
ment about each Haversian canal (Fig. 35, b). But it will also
be seen that there are other groups of lamellae whose arrange-
ment is slightly different. For example, at the periphery of
the bone their direction is parallel with the surface.
Such lamellae may be represented at a. They are known as
the intermediate or circumferential (Tomes and De Morgan).
Another group, only partly encircling each canal, is known as
the peripJieric or interstitial, c. The first mentioned, imme-
diately about the canal, are the concentric, b.
Schaefer believes with Sharpey that each lamella consists of fibres crossing
each other diagonally, and separated on either side by a homogeneons layer.
According to Von Ebner, the peculiar cross striations belong only to Canada
balsam preparations that are old. These markings are due to the peculiar
refractive power of the balsam which fills the canaliculi.
THE CONNECTIVE SUBSTANCE GKOUP.
91
The arrangement just described is found in all compact
bone where there is any considerable thickness, but when, as
in flat bones, the cortex is very thin, the lamellae often pursue
a straight arid parallel course. Some of these lamellae or plates
exhibit transverse striations ; others are homogeneous.
In Fig. 35 may be seen the lacunae lying between the
lamellae. They appear
as dark spaces disposed
at quite regular intervals
and, having their long
axes parallel with the
course of the lamellae.
Laterally each corpuscle
gives off numbers of pro-
cesses, many of which
branch, while all, or near-
ly all, anastomose with
corresponding branch-
lets of other corpuscles-
A branchlet is also given
off from the end of each
corpuscle, and forms a
connection with the adjacent bodies lying in the same inter-
lamellar space and in the same plane.
The Haversian canals form a broad-meshed network through-
out the bone, establishing a communication between the central
marrow cavity and the external surface of the bone (Fig. 36).
The arrangement of parts comprised by each Haversian
canal, with its investing lamellae, and interposed lacunae and
their anastomosing carialiculi constitutes an Haver sian system.
Though found mainly in the compact tissue, they may also be
seen in the large trabeculae of the spongy substance. As seen
in Fig. 36, the Haversian canals form a network of which the
longitudinal tubes are the larger and longer. Besides convey-
ing blood-vessels and lymphatics they have a certain amount
of connective tissue which varies according to the locality, and
establishes a more or less complete connection between the con-
nective tissue of the marrow cavity and of the periosteum.
In young bone this is well seen ; in adult bone the direct
continuity can with difficulty be traced, as the vessels are apt
to till the tubes pretty completely.
PIG. 35. Transverse section of human femur, deprived of
inorganic material by hydrochloric acid. (Rollett.)
92
MANUAL OF HISTOLOGY.
Preparation of dry bone. In order to study the char-
acteristics which have just been described, any human long
bone may be taken. It should be stripped of its soft parts,
bleached, and well dried. Thin sections are then to be made
both in a longitudinal and transverse direction, with a watch-
spring saw.
Next, cleanse them well in water to which a little bicar-
bonate of soda has been added ; then place on a whetstone
and grind down by rubbing backward and forward with the
finger until they are suffi-
ciently thin ; or the sections
may be placed between two
plates of ground glass and
rubbed down.
Finally, when so thin that
type may be read through
them, mount either dry or
in Canada balsam or dam-
mar varnish. All the char-
acteristics already described
may then be seen.
Preparation of decalci-
fied bone. Another method
consists in first removing the
earthy salts. If it is desira-
ble to accomplish the work
rapidly, cut the bone to be
prepared into the smallest
available pieces and immerse
from four to five days in a 10
per cent, watery solution of
Fio. 36. -Longitudinal section of human nlna, show- hydrochloric acid,
ing the Ha versian canals forming meshes. (Rollett.) m , , ,. ,, .
The completion of this
process may be determined by testing the bone with a fine cam-
bric needle. So long as it meets with resistance, the presence
of the bone-earths is certain ; on the other hand, if it enter
easily, the process of decalcification is over, and the piece ready
for cutting.
Now wash thoroughly in water, so as to remove the acid,
place in 80 per cent, alcohol, gradually increasing the strength
to 95 per cent. The specimen is then ready for use and may
THE CONNECTIVE SUBSTANCE GROUP. 93
be treated precisely as any other tissue of the body. If more
time is at the disposal of the student, chromic acid may be
used in a J per cent, solution. This process is rather slow, re-
quiring several months. It may be materially hastened by the
use of nitric acid (2 per cent.). It has been found that after
immersion in chromic acid for a few days, the soft parts are
rendered insensible to the action of other strong acids, such as
nitric and hydrochloric, when used in the dilute form. These
chromic acid preparations are exceedingly beautiful objects
FIG. 37. Bone lacunaa with their processes. (Rollett. )
when seen with low powers. The matrix is of a deep grass
green. If a thin section is stained with borax-carmine (Arnold's
formula) the bone-corpuscles and connective tissue are stained
red, and the contrast of color brings out the finer elements
very distinctly.
Picro-carmine may also be used, and then the muscular tis-
sue, if any chance to adhere to the bone, is stained yellow ; or
eosine and hsematoxylin may be used instead of borax car-
mine, and thus very excellent examples of triple staining pro-
cured. Sometimes a saturated solution of picric acid is em-
ployed to decalcify, but the excess of acid, after taking out
the bone-earths should be thoroughly removed by soaking in
94 MANUAL OF HISTOLOGY.
water before immersion in any staining fluid. In preparing a
specimen for cutting with the knife it may conveniently be
held in the hand, or, if the microtome is used, the bone may
be embedded in the ordinary mixture of wax and oil, pith, or
liver, according to methods already described. Rutherford re-
commends glycerine jelly for this purpose.
Any of these plans of preparing decalcified bone will reveal
the presence of the bone-corpuscles within the lacunae. These
will be found to correspond quite closely in size and shape
with the cavities. They may also be shown to have a direct
continuity with the connective- tissue corpuscles of the perios-
teum. In growing bone this is more evident. A nucleus can
also sometimes be seen in the bone-corpuscle. In Fig. 36
the lacunae, with their canaliculi, are well shown.
Sharpens perforating fibres. Attached to the outer sur-
face of compact tissue, and penetrating the bone at right an-
gles, are certain fibres which have been named after Sharpey,
their discoverer.
Take a flat bone of the skull that has been decalcified, seize
pieces with the forceps, tear them out from the surface, and
examine in water. In some of the fragments the bundles of
fibres will be seen ; in others the lamellae, perforated for the
fibres. If a portion of tendon adhere to the bone, and a sec-
tion be made through the two at their line of apparent junc-
tion, it will be seen that the tendon-fibres are continuous in
the bone with Sharpey' s fibres.
A very prevalent view is that they constitute the remains of
the periosteal processes, which we shall see are largely con-
cerned with the ultimate development of bone.
Cancellous tissue. All of the elements of bone, that go to
make up a Haversian system, are found in the cancellous
tissue, so that, in this respect, it does not differ from the com-
pact. The chief peculiarity lies in the marrow cavities, or
channels, as they might appropriately be called, and they indi-
cate either, on the one hand, that the bone is passing through
a developmental stage ; or that it is being rarefied by a process
of retrograde metamorphosis ; or, finally, that it has reached a
stadium of repose in either of the first-named changes. These
points will be further particularized when the growth and de-
velopment of bone is explained, but the reader is now prepared
THE CONNECTIVE SUBSTANCE GEOUP. 95
for the rather remarkable proposition that compact bone is
formed out of spongy, and spongy out of compact.
These marrow channels are a series of branching and anasto-
mosing tubes, rich in corpuscular elements and vessels. In
young bone the latter are known as red marrow. When a
longitudinal section has been made through a tubular bone, it
will be seen that the channels are enclosed in an osseous net-
work, whose meshes differ much in shape. In the articular
extremities they are long and narrow ; at other points, more
nearly quadrilateral.
There is a second variety of marrow, known as yellow, which
is found in the central cavity of the long bones. The yellow
color is due to the presence of fat, though it also contains
peculiar, small, colorless corpuscles, not unlike the leucocytes
of the blood, and known as marrow-cells, together with the
ordinary branched and nucleated connective-tissue corpuscles,
also large multi-nucleated bodies that are usually granular and
sometimes striated, and blood-vessels. The large corpuscles
are the myeloplaxes of Robin (giant-cells).
The red marrow also contains marrow-cells, though but few
fat-cells. It is remarkable for being the seat of the peculiar
nucleated blood-corpuscles that have been described by Neu-
mann and Bizzozero. They are transitional between the white
and the red in size, and have a uniform yellowish green color
(Klein).
The authors above referred to found the nucleated corpuscles in the red
marrow of the ribs and bodies of the vertebra ; they resembled blood-corpus-
cles that are found in the human embryo, and were regarded as evidence that
the bones have bloodmaking properties. Later researches (Orth and Litten)
have seemed to corroborate these views, and to have shown that in certain
morbid states of the blood, as in carcinoma, phthisis, and syphilis, an effort of
this kind is made for the relief of the constitutional infection. Experiments
upon dogs have also added further testimony and have shown that after extreme
artificial anaemia there is a new formation of blood-globules, in which the
nucleated bodies play an active part, together with other elements, such as the
giant-corpuscles of Hayem, etc. These views, however, have met with opposi-
tion, and Eutherford (" Pract. Histology," p. 88) maintains that the nucleated
corpuscle is an indication of corpuscular disintegration rather than of new-
formation.
The periosteum is a layer of dense fibrous tissue closely
covering the bone, and connected with it by a thinner layer of
96 MANUAL OF HISTOLOGY.
looser texture. The external portion may be composed of sin-
gle, double, or treble laminae of varying thickness. The inner
or osteogenetic portion is of great interest and importance,
as it contains the osteoblasts, which are active agents in the
formation of a great part of all bones, as we shall presently
see.
Development of bone. Views as to the method by which bone
is formed have undergone great changes within the past few
years, and it may be stated that most modern observers have
given in their adhesion to the theory that bone is not developed
by a calcification of cartilage, but by a long and complicated
series of changes inaugurated by the corpuscles of the marrow
cavities, on the one hand, and those of the periosteum, on the
other. These conclusions have been the result of very extended
researches conducted by a variety of methods and upon many
kinds of animals.
As the mode of growth in man and horned cattle is identical,
a good method of procedure is as follows. Take the hoof of a
yearling bullock, and, removing the bones, macerate them a
few days in a 10 per cent, watery solution of h3 7 drochloric acid
and then in chromic acid (gr. ij. lj.). In a few days they
will be decalcified sufficiently to allow of a thin section being
shaved off from the surface so as to include parts where ossifi-
cation has already commenced. The sections may then be
stained in a neutral solution of carmine and mounted. The
gradual stages between the advancing bone and the liquefying
cartilage can now be studied. Following the changes from the
surface of the articulation toward the centre of the bone, there
is seen at first, beneath the fibrous layer, a stratum of hyaline
cartilage. The corpuscles are long, flattened, and lie parallel
with the surface. Passing to a greater depth they become
larger, and increase in number by gradual progression. As
these capsules enlarge and their contents multiply, they
begin to be arranged about the wall of the cavity, while
the matrix gradually wastes away. A little farther and
there is a deposit of calcific material in the intercapsular sub-
stance. Another step internally and the cartilage capsules
have in part coalesced, and now they are beginning to be filled
by the marrow tissue pushing up from the central parts of the
bone. When the connective tissues and vessels that constitute
this arborescent growth have entered the capsules, the corpus-
THE CONNECTIVE SUBSTANCE GROUP. 97
cles that line them are called osteoblasts. Whether or not they
are identical with the cartilage-corpuscles, or belong to the
budding marrow-processes, seems to be a matter of doubt.
Klein intimates that the cartilage-corpuscles disintegrate. Ran-
vier has seen no proof of it. It is probable that some of the
cartilage-corpuscles persist, certainly to a limited extent,
and preside over the remains of the calcified cartilage. The
bulk of the new bone is made up, however, of new material
which is deposited under the form of concentric lamellae about
the marrow cavities, most likely by a proliferation of the
osteoblasts.
These changes may all be observed to advantage in the
specimen just mentioned, and the successive gradations of the
process can be conveniently magnified, so as to be easily seen,
by making sections obliquely to the surface of the bone. With
a low power the specimens will have uncommon beauty, as the
corpuscles take the carmine well, while the interstitial tissue is
of a bright, transparent grass-green.
In a vertical section of a long bone, while the process is
essentially the same, there are some modifications in the suc-
cessive steps. Thus the spongy bone of the epiphysis en-
croaches on the cartilage, causing it to be absorbed in the man-
ner already described, but the intermediary cartilage, lying
between the epiphysis and diaphysis, is seen to have its cor-
puscles arranged in long lines parallel with the axis of the