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Studies of cartilage and osteoid arising spontaneously and experimental attempts to induce their formation in ear chambers.

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Studies of Cartilage and Osteoid Arising
Spontaneously and Experimental Attempts
to Induce their Formation in Ear Chambers’
ROY G. WILLIAMS
Department of Anatomy, School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania
-
ABSTRACT
Clark and Clark using a rabbit’s ear chamber in which the observation space was in direct continuity with perichondrium reported cartilage arising
spontaneously in more than 40% of chambers. With a chamber of different design
having a very restricted relationship to perichondrium, osteoid, that is, an unmineralized bone-like tissue, appeared i n 5 of 110 animals and in two of the five there were
also unrelated islands of cartilage. In one additional animal osteoid developed three
months after local application of continuous pressure of minute degree. In the remaining 104 animals no cartilage or osteoid formed irrespective of the experimental
usage to which the chambers were put. The results are interpreted to mean that in
rabbit’s ear chambers cartilage or osteoid may form from precartilaginous cells resident
in or derived from perichondrium but not by metaplasia of morphologically similar
connective tissue cells. If a special stimulus was necessary to activiate the precartilaginous cells then the only obvious ones were friction and pressure both of very
small degree but continuously maintained for long periods.
Bone and cartilage sometimes appear
spontaneously in the tissue that invades
transparent chambers installed in rabbit’s
ears. This was first reported by Clark and
Clark (’42). They observed cartilage formation in 10 of 23 chambers studied for
from 4 to 20 months. Occasionally bone
formed in the cartilage. Cartilage first appeared at various times after chambers
were vascularized, mostly within from two
to four months and never in less than two
months. It arose from elongated motile
cells containing uniformly distributed
granules. The cells became stationary and
spherical and the granules enlarged and
coalesced to form large fat droplets. At
the same time a clear homogeneous substance appeared between the cells. Elongated cells at the periphery underwent
similar transformation thereby increasing
the cartilage mass. Increase in size was
limited and not steadily progressive and
occasionally small masses underwent regression. It seemed evident to Clark and
Clark, “that some special localized chemical condition of the tissue must serve as
a stimulus for the metaplasia of connective tissue cells or the differentiation of
specific precartilage cells into cartilage in
the chamber area, in view of the sporadic
and restricted formation of cartilage.”
Clark and Clark reported no bone that
arose spontaneously unrelated to cartilage.
However, in one specimen they observed
bone formation in small cartilaginous
masses in the observation area and concluded that it was produced by metaplasia
of cartilage cells. They stated that the
bone so formed was true bone but did not
mention whether or not it was mineralized.
The data reported here are summarized
from studies of 110 chambers used for a
variety of purposes over a period of about
ten years. The chamber, one per rabbit,
differed in its construction and relationships to the tissues of the ear from the
type used by Clark and Clark. All chambers were studied for not less than one
and no more than four years with the
average at about two years. A much larger
number of chambers were excluded from
the series because they were studied for
less than one year. Reason for the exclusion was that Clark and Clark reported
one instance in which cartilage appeared
11 months after complete vascularization.
No cartilage or osteoid arose in any of the
excluded specimens. As will be noted
hereafter none of the spontaneously arising bone-like masses was mineralized. For
that reason they will be designated as
osteoid rather than bone.
1 This investigation was supported by a research
grant from the National Science Foundatlon.
93
94
ROY G. WILLIAMS
Spontaneously arising osteoid was seen
in only 5 of the 110 animals and in two
of the five there were also small unrelated
islands of cartilage. No bony transfonnation in the cartilage was observed. In one
additional animal osteoid appeared following an experimental procedure. No bone,
osteoid or cartilage appeared in the remaining 104 chambers irrespective of the
usage to which they were put. This is in
contrast to the more than 40% of chambers in which cartilage developed spontaneously as reported by Clark and Clark. Differences in chamber design and relationship to the tissues of the ear may well have
been responsible for differences in results
obtained by Clark and Clark and those
reported here. The significance of the differences may be to point to the nature of
the stimulus that caused the formations as
well as the source of the cells involved.
The chamber used by Clark and Clark
is illustrated in cross section, diagrammatically and not to scale, in A figure 1. It is
called the “round table” type to distinguish
it from others. It was described by Clark,
Kirby-Smith, Rex and Williams (’30). The
central clear area is the observation space
which vessels and connective tissue invade and where the cartilage developed.
It has plastic material, either Lucite or
Kodaloid, on one side and mica on the
opposite. When installed, the circular observation space is bounded laterally by the
original cartilage and perichondrium of
A
Mico
&
_*
PIostiC
I
‘c
“ k,,&d
Calf.rdglge
B
M~co
-
Tantalum
Tantalum gauze
subcutaneous tissue
Inner skin
Paper
Lucite
Other melols
Fig. 1 Cross section diagrams of two ear
chambers, natural size but not to scale, showing
their parts and relationships to the tissues of
the ear. A is the “round table” type used by
Clark and Clark. B is the tantalum and mica
type used for studies reported herein.
the ear. This type cannot be used successfully without protection when not being
studied since it cannot withstand the animal‘s scratching and other attempts to
remove it. Protection is provided by
splints and removable shields covering the
chamber and nowhere connected with it.
Movement of the device in the ear is resisted by friction on tissue between upper
and lower elements, by the central table
which extends through a hole in the cartilage and by three bolts that hold the chamber together and extend through the entire thickness of the ear. There is always
some tendency for movement toward the
tip of the ear since with the rather heavy
splints and shields used for protection the
ear hangs down when the specimen is not
being studied and displacement would
tend to be from a thicker toward a thinner
part of the ear. The motion is very slight
and can be detected only by slow shift in
position of microscopic landmarks over
many weeks. Chambers of this sort weigh
3.5 gm exclusive of devices used for external protection. Such drag on the contained tissue as the weight of the chamber
produces is continuous when the animal
is in normal sitting position. The movement is not extensive or rapid enough to
cause sticking of leucocytes to capillary
walls, a delicate sign of vascular injury,
but it may visibly S e c t the orientation of
connective tissue particularly around the
buffers that control the thickness of the
observation space.
The type of chamber used for the studies
reported here is illustrated diagrammatically in cross section at B, figure 1. With
all parts in place it weighs 3.9 gm. When
installed the original cartilage of the ear
is nowhere closely related to the observation space as it is in A, figure 1, and the
perichondrium has only a limited and indirect relationship. The observation space
is bounded above and below by mica and
laterally by tantalum and mica or in some
instances by mica only. It was described
by Williams and Roberts (’50) and in a
modified version by Williams (’61 ). Movement in the ear is resisted by firm compression of the cartilage when the chamber is
‘installed. When it becomes stabilized,
connective tissue and vessels extend
through the meshes of the metal gauze
CARTILAGE AND OSTEOID
from side to side and movement is then
further resisted by a large number of connective tissue pillars extending through
the chamber itself. No external protection
is necessary and when not being studied
the ear is held in its normal position.
Movement of this type chamber in an ear
cannot be detected by any change in position of tissue landmarks. Thus, the contents are not subjected to the same degree
of displacing force or tensions that more
obvious movements would entail. It should
be emphasized that in any case with any
chamber in an ear the force applied to the
contents as a result of the weight of the
device are very slight but such as there
may be operates continuously when the
animal is in normal sitting position.
The chamber illustrated at A, figure 1
can readily be taken apart and removed
since its parts are solid, the surfaces
smooth and tissues do not adhere to it.
Clark and Clark reported that bone frequently developed around the bolt holes
in areas that could not be seen until the
chamber was removed. The chamber
shown at B, figure 1 can be removed only
by sharp dissection. In no case in which
that was done was bone or cartilage found
in regions beyond the observation space.
When a chamber is installed too close to
the fold of the ear or if the ear is not as
wide as i t should be to accommodate the
chamber, connective tissue overgrowth outside the chamber sometimes forms where,
in such cases, the chamber impinges on
the fold. These growths have no effect on
the chamber contents but may become
large enough to require excision which
may have to be done several times in the
course of two of three years. Since slight
mechanical irritation was apparently the
stimulus for the overgrowths, and since
the stimulus was applied continuously for
many months, the pieces excised were
routinely sectioned with the thought in
mind that they might eventually become
malignant. They did not even in cases
studied for as long as four years which is
well past the midpoint of a rabbit’s life
expectancy. Of all such excised pieces
from all animals none contained bone or
osteoid. Only one contained cartilage and
it was connected to the original cartilage
of the ear. This piece was the first excision
95
from the animal. Subsequent excisions
from the same animal were composed entirely of fibrous tissue as were those from
other animals. Local mild mechanical irritation applied continuously for many
months did not result in ectopic bone or
osteoicl formation in fibrous tissue outside
the chamber which responded to continuous injury by proliferation and no cartilage formed unrelated to the original ear
cartilage.
S m e properties of spontaneously
arising cartilage and
ectopic osteoid
In none of the five chambers in which
osteoid or cartiIage appeared spontaneously were the first stages of development
observed. This was because the animals
were prepared for other purposes and until
osteoicl was seen they were not studied at
frequent intervals since they were held as
stock animals. However, it was determined that no osteoid or cartilage appeared spontaneously earlier than two
months or later than five months after
complete vascularization of the chamber.
When first seen the ectopic osteoid consisted of separate discrete masses, one to
three per chamber. They were irregular
in shape and not encapsulated. In the
two chambers containing cartilage as well
as osteoid there were one and two small
masses respectively, all unrelated to the
osteoid. They were not vascularized and
underwent no observed changes.
The smallest osteoid mass was .15 mm
X .3 mm in surface diameter and the
largest, (fig. 2), measured 1.5 mm X
.6 mm. They were the same thickness as
surrounding tissue ,c‘ ,y ’ they were a
part. Occasional vessels . tended over or
under them but not through or in them.
These vessels were less numerous than
they would have been in an area of connective tissue of corresponding size. One
got the impression that there were very
slow and slight changes in outline of the
osteoid masses when observed daily over
many weeks but measurements of greatest
and least diameters made on enlarged serial photomicrographs showed no change.
Figure 16 is a photomicrograph of cells
near the center of the mass illustrated in
figure 2. It was taken while the piece was
96
ROY G . WILLIAMS
in the living animal. Lacunae appear
black, unlike those in mineralized bone
growing from autografts of cancelous
bone, (compare fig. 16 with figs. 18 and
1 9 ) . Canaliculi radiate from lacunae and
are coarser and fewer in number than
those seen in living osteocytes of bone
autografts.
All pieces of spontaneously arising osteoid were eventually removed for sectioning. They were fixed in 10% neutral formalin, embedded in paraffin and cut at
10 N. No difficulties were experienced in
cutting them and no parts of sections
blackened with silver by the von Kossa
method. Therefore, the pieces were not
mineralized and all were osteoid rather
than true bone. Sections prepared with
Schmorl's method demonstrated cells resembling osteocytes but cell processes
were larger, uneven in length, fewer in
number and less evenly distributed.
Electronmicrographs of thin sections of
appropriately fixed and embedded blocks
of osteoid were kindly made by Mrs. Robert Eager and examined by Dr. Frank A.
Pepe. Collagen fibers were present but no
mineral crystals. Distributed among the
cells were a few large black irregular
shaped masses not seen in control sections of cancellous bone from the ilium.
These were interpreted as dead cells. They
were not sufficiently numerous to suggest
that the entire osteoid mass might have
been dead, or dying.
To determine reaction of the osteoid to
injury the experiment illustrated in figures
2 to 7 was done. The cover of the chamber was removed under fluid and the tip
of the piece cut off, reversed and transplanted in the same chamber well away
from its original site. The cover was then
replaced and observations made for many
weeks thereafter. The transplanted piece
was laid on the vessels at its new location.
Except for very slight alteration in outline
it underwent no changes, stimulated no
vessel formation or leucocyte infiltration
and was unaffected by the close presence
of the relatively large vein that formed
around one end, (figs. 6 and 7). It is
illustrated at higher magnification in
figure 15.
The hole left by the excision gradually
filled in with connective tissue and blood
vessels. In making the excision a small
fragment of osteoid was dislodged and
left near the cut edge of the main piece.
This is illustrated by arrows in figures 4,
5 and 6. The cut edge of the main mass
rounded somewhat as healing progressed
but the small piece nearby increased
slightly in size, changed in shape and
finally fused with the main piece, (figs. 4,
5 and 6). The piece after fusion is illustrated at higher magnification in figure 14.
From the time of operation until fusion
about two months elapsed. How the fusion
was produced was not observed; in fact,
changes were not noticed at all until photographs taken subsequent to operation
were arranged serially. It is considered
likely that the process was on a smaller
scale similar to that illustrated in figures
8 to 13 inclusive and figure 17.
The central black spot in figure 8 represents beginning of growth in the remains
of an autograft of cancellous bone. Over
a period of about five months it slowly increased in size by addition of successive
layers of spindle-shaped cells surrounded
by amorphous matrix. The piece was on
the host tissue but not part of it. It had
no blood supply of its own and did not
alter the arterial pattern of the host area
it covered. Figure 17 is a higher magnification of the notch at the upper margin
of the mass in figure 12. It illustrates the
laminated arrangement of spindle-shaped
cells and, at the arrow, a new layer forming. This piece when removed and sectioned demonstrated the same structurr
as those pieces that arose spontaneously
It was not mineralized and therefore osteoid rather than true bone. The mass
before removal, as shown in figures 8 and
9, is black because the cells are close to
gether. In figures 10 to 1 3 the mass is
less black possibly as a result of spreading
of the cells by interstitial growth although
in tissue of this sort it is generally supposed that interstitial growth does not
occur. In photomicrographs of osteoid in
the living animal, lacunae and canalicul
always appear dense black unlike those
in living autografts of cancellous bc ne.
Reasons for this are obscure but it is not
because they are empty.
The mass illustrated in figures 8 to 13
is the only instance of its kind encoun-
CARTILAGE AND OSTISOID
tered in a large number of autografts of
cancellous bone, that is, it behaved like
spontaneously arising osteoid. All other
cancellous autografts either grew vigorously or underwent rather rapid resorption, tke proportion being about 50-50,
(Williams, '62). In contrast with the result of slow appositional growth, figure 17,
with no localized resorption, figures 18 and
19 show the type of growth change seen
at the periphery of an autograft of cancellous bone that had true osteogenic potencies. Figure 18 illustrates the peripheral osteogenic fibrous layer, between the
arrows, in a dormant state. WLen growth
begins this changes as shown in figure 19
and mineralized bone is laid down beginning adjacent to the older bone and extending gradually outward. The growth
appears to be a product of graft cells only.
Subseqcent history of such autografts consists of small internal resorptions and replacements (Williams, '57).
In one of the five chambers that
contained spontaneo..sly arising osteoid
masses three autografts of cancellous bone
from t: e ilil m were made. Grafts were
about the same size as the osteoid. One
graft underwent resorption and two exhibited extensive growth after the manner
illustrated in figures 18 and 19 and as
previously described (Williams, '57). During growth of the autografts the osteoid
masses were slowly covered and the ultimate fate of the osteoid could not be determined, but while being covered i t underwent no changes. This experiment was
done to investigate whether the limited
growth characteristics of the osteoid could
be attributed to vascular deficiency or
some other inadequacy of the environment
3r to limited capacity of the osteoid cells
khemselves. The results indicated that the
osteoid was deficient since the environment and vascular responses were adelluate to support true bone formation in
cells that had that potential.
Experiments with ear chambers
The transparent chamber method has
eel's used in this laboratory of late years
chietfy to study the survivability of autografts and homog-rafts and to investigate
the histophysiology of those tissues that
survived indefinitely. Small autografts
I
97
that survive indefhitely under such circumstances are regarded as microcosms
that reflect the structure and activity of
parts not otherwise accessible for repeated
and prolonged microscopic study in living
mammals. With the exception of grafts
of bone and of marrow from which bone
formed, no cartilage, bone, or osteoid appeared in any chamber after a grafting
operation irrespective of the tissue grafted
and irrespective of whether or not the
grafts survived or whether they were auto
or komografts. This is in contrast to the
anterior chamber of the eye or brain
wherein grafts of non-skeletal tissue sometimes produce bone.
Among the 25 tissues studied as autografis skeletal muscle was included. Two
small strips from the medial edge of a
sternothyroid muscle were autografted in
each of three animals. Grafts were about
1 mm X .5 mm and of undetermined
thickness. They demonstrated cross striation from the beginning and became vascularized in a manner characteristic for
skeletal muscle. The fibers underwent
rhythmic21 contractions but only when
stimulzted by bright light. Contraction
rates differed in different fibers of the
same graft and may have been fibrillations
rather than true contractions. After about
six months the striated fibers slowly disappeared and were replaced by connective
tissue but no bone or cartilage formed.
In investigating the behavior, appearance and properties of tissue in chambers
it has been injured repeatedly or killed in
small areas by cutting or bruising, by
slow and by rapid changes in internal
pressure, by exposure to ultraviolet light,
by injection of vital dyes, by local infection both acute and chronic and by intermittent anoxemia of long duration. Also,
various kinds of foreign bodies have been
implanted including calcium salts and
chemical carcinogens. After each procedure the specimens were followed microscopically for many months. In none of
these studies did bone, cartilage or osteoid
form. The experiments with anoxemia
were done in the following manner: pressure was applied to the flexible covers of
two chambers by U-shaped screw clamps
capable of delicate adjustment. The clamps
were constructed so that they did not in-
98
ROY G. WILLIAMS
terfere with microscopic observations. Sufficient pressure was slowly applied to
press out the blood and just stop all blood
flow. The tissue was slowly acclimated to
this treatment over several days until the
periods of anoxia were for from four to
six hours duration. This was repeated
daily five days per week for seven months.
The experiment was an attempt to repeat
in living animals the experiments of Goldblatt and Cameron ('53) on tissue cultures wherein fibroblasts became malignant after repeated exposures to anoxia.
The results of my studies were striking
but no malignancy developed and also no
bone or cartilage formed although that
might have been expected if interference
of that sort regularly induced metaplasia
in connective tissue or activated precartilaginous cells.
The single instance in which osteoid
formed following an experimental procedure occurred after attempts to determine
oxygen tension at different parts of
the microcirculation using microelectrodes
constructed as described by Davies and
Brink ('42). The mica cover of a chamber
was replaced with two covers, the inner
one, about 75 IL thick, having four holes
drilled in it each about .5 mm in diameter.
When the disturbance associated with replacing the cover subsided, the outer cover
was removed under fluid thus exposing
vessels under the holes in the remaining
cover. With a manipulator a microelectrode was then placed successively on different parts of the vessels exposed without
stopping flow. Vessels under such small
holes are not affected by removal of the
outer cover. This procedure was not successful as a means of measuring oxygen
tension since the vessels proved to be insulated by a thin layer of connective tissue. The outer cover was then replaced.
Some time later it was observed that tufts
of capillaries and connective tissue filled
all the holes in the inner cover. To reclaim the specimen for other use both
covers were removed, about half the tissue
that had been in each hole was cut away
and a single cover with no holes in it was
applied with enough pressure to flatten the
tissue projections but not enough to stop
circulation in capillaries at their bases.
The animal was then placed among stock
animals held for projected studies. A single osteoid mass appeared three months
later under one of the four sites where
newly formed connective tissue had been
locally subjected for a long period to continuous pressure of slight degree. The
mass was in all respects similar to those
previously described that arose spontaneously. This procedure was repeated in
another animal but no osteoid formed.
DISCUSSION
The tissue that invades the observation
space in a transparent chamber installed
in a rabbit's ear consists of fibroblasts,
blood vessels and various other cell types
generally found in connective tissue including lymphatic vessels and nerves. In
the majority of cases cartilage, osteoid or
bone never appear in it. However, if the
observation space is in direct continuity
with the surrounding perichondrium and
cartilage, invading tissue will eventually
have cartilage in it in about 40% of cases
as reported by Clark and Clark ('42). If
the chamber is so designed that the observation space has no relationship to the
ear cartilage and only a limited and indirect relationship to the perichondrium
cartilage appears in the invading tissue
in less than 2% of cases as reported here.
This suggests that the cells involved in
cartilage formation were carried in from
the perichondrium with the invading tissue and, although indistinguishable in the
beginning from ordinary connective tissue
cells, were specific precartilaginous cells
rather than cells formed from some other
type by metaplasia. Cartilage might foni.
from undifferentiated mesenchymal cell
resident in connective tissue but there ic
no reason to suppose that there were an)
more such cells in Clark and Clark's preparations than there were in mine if indeed
they were present at all.
In no case was cartilage identifiable
within two months of the time chamberc.
were completely vascularized. It generallj
appeared between the second and fifth
months although Clark and Clark reported
one case in which it appeared as late as
eleven months. This delay in development
may have been nothing more than an
expression of the time required for cartilage to mature from progenitor cells or it
-
CARTILAGE A N D OSTEOID
may mean that a particular type of stimulus was required or that continuous application of a stimulus for relatively long
periods was essential before precartilaginous cells exercised their special potentialities. Rodbard ('58) produced hyaline cartilage in an arterial wall by reducing the
distensibility of a segment of the wall by
implanting a steel wire through the lumen.
He explained the result as a metamorphosis of connective tissue in response to a
high rate of compression resulting from
the pulse wave hammering against tissue
of the wall held in position firmly by the
wire.
Clark and Clark ('42) thought it was
obvious that some localized chemical condition of the tissue in chambers must have
been the stimulus that induced metaplasia
or activated precartilaginous cells. There
is no reason to suppose that the chemical
composition of tissue in their chambers
was any different from that in the chambers considered here unless thin tissue
with plastic on one side and mica on the
other is, because of the plastic, different
in some way from tissue that has mica on
both sides.
If a special stimulus was necessary to
start the cartilage formation in chambers
then the most obvious factors that might
have been stimuli were those associated
with tendency of the chamber to migrate
as a result of its weight. In the type that
permitted some movement cartilage formation was common and in the type where
movement was imperceptible it was uncommon. However, movement of the
chamber with consequent friction on its
contents could be convincingly evoked as
the stimulus only if the observation space
of all chambers had the same relationship
to perichondrium. Continuous friction of
small degree must be considered as a possible stimulus for cartilage formation under the circumstances given here but at
present it is hypothetical.
Clirk and Clark ('42) observed the
fhst formation of cartilage and in some
,:,' e5 its transformation to bone. First
:.,
s of cartilage or osteoid formation
w+1'6' not seen in my preparations for the
n 3 .:n given and hence it is not known
1 11, ?er or not the osteoid was preceded
'J,
Lrtilage. There are no clear indicaI
~
7
99
tions as to why the osteoid formed but it
seems likely that the cells involved were
originally derived from perichondrium.
The osteoid, unlike the cartilage, did have
a very limited ability to slowly increase its
mass by addition at the periphery of spindle shaped cells surrounded by amorphous
matrix. Failure of the osteoid to mineralize was a function of its cells and not the
result of any inadequacy of the environment, vascular or otherwise, since autografts of cancellous bone in the same environment grew vigorously and were mineralized.
Only a single instance of osteoid formation following an experimental procedure
was encountered. In this chamber four
small sites, all similar in their cellular
composition and size, were subjected simultaneously to the same continuous pressure of very small degree. After an interval of about three months osteoid appeared
in one of the sites. This was repeated in
another animal but no osteoid formed.
The osteoid in this instance is attributed
to the chance presence in the area of cells
from the perichondrium. If a special stimulus was necessary to activate the cells
then the only obvious one was the continuously maintained pressure which was
at no time sufficient to collapse capillaries
in the neighborhood.
With the exception of the instance mentioned above, no cartilage or osteoid
formed in the tissue filling the observation
space irrespective of the duration, type or
amount of experimental injury to which
the tissue was subjected. This suggests
that either none of the stimuli were adequate to induce cartilage or osteoid formation or there were no cells present capable
of responding.
With respect to grafts in chambers, except for some of skeletal tissue from which
bone formed, no cartilage, osteoid or bone
formed in or in relation to any graft irrespective of the source, type or fate of the
graft cells. In other areas of the body,
grafts have been reported to produce ectopic bone or cartilage by metaplasia
(Bridges, '59) although Danis ('59) found
no evidence that skeletal grafts induced
mataplasia in host cells. The failure to
induce cartilage or osteoid experimentally
together with other observations reported
100
ROY G . WILLIAMS
here suggest that when cartilage or osteoid
appear in an ear chamber the responsible
cells are predetermined cells from the perichondrium and that metaplasia of common connective tissue cells as a source of
cartilage or osteoid must be rare if it occurs at all in the ear.
Ham "30) is of the opinion that osteogenic cells have a dual potentiality. In a
vascular environment they form bone and
in a nonvascular environment they form
cartilage. If they differentiate in an environment that is neither one nor the other,
they form a tissue that has some characteristics of both bone and cartilage. Clark
and Clark ('42) observed that cessation of
growth and retraction of vessels followed
cartilage formation but if bone formed in
the cartilage vessels invaded it. In the
growth of bone from cancellous autografts
beginning of growth preceded endothelial
proliferation, (Williams, '62) and if bone
growth halted so did the formation of
new capillaries. Extent of vascularization
with consequent effect on local oxygen
availability may well have the importance
attributed to it by Ham as far as an exciting stimulus for the formation of cartilage
or bone from a fibroblast-like cell is concerned. However, once formed the cartilage or bone would seem to regulate its
vascularity and not the reverse.
As to why osteoid formed nothing much
can be said unless continuous and long
maintained pressure of small degree was
of importance in activating certain cells
initially indistinguishable from fibroblasts.
Rodbard ('62) reported that when a segment of blood vessel is subjected to continuous compression, osteoid or calcific
changes or both may occur.
The data presented here are interpreted
to mean that in the rabbit's ear there is a
race of fibroblast-like cells whose destiny
it is to form cartilage or osteoid. Other
fibroblast-like cells in the ear morphologically indistinguishable from precartilaginous cells were not induced to form cartilage by any of a variety of experimental
means. Osteoid, so called here because it
had some cellular characteristics resem-
bling bone but was not mineralized,
formed occasionally from precartilaginous
cells or by transformation of cartilage. The
only obvious things that might have
caused or contributed to the activation of
precartilaginous cells were friction and
pressure together or separately, both of
slight degree but continuously maintained.
LITERATURE CITED
Bridges, J. B. 1959 Experimental heterotopic
ossification. International Review of Cytology,
ed. by G. H. Bourne and J. F. Danielli. Academic Press, N. Y. Vol. 8, pp. 253-278.
Clark, E. R., and E. L. Clark 1942 Microscopic
observations on new formation of cartilage and
bone in the living mammal. Am. J. Anat.,
70: 167-200.
Clark, E. R., H. T. Kirby Smith, R. 0. Rex and
R. G. Williams 1930 Recent modifications in
the method of studying living cells and tissues
in transparent chambers inserted in the rabbit's ear. Anat. Rec., 47: 187-211.
Danis, A. 1959 In an osteogenic skeletal graft,
the new-formed bone develops differently according to its autologous or homologous origin.
Biological Problems of Grafting, A symposium
pp. 447453, P. B. Medawar, Chairman, Charles C Thomas, Springfield, Ill.
Davies, P. W., and Frank Brink, Jr. 1942 Microelectrodes for measuring local oxygen tension i n animal tissues. Rev. Sci. Instr., 13:
524-533.
Goldblatt, H., and G. Cameron 1953 Induced
malignancy i n cells from rat myocardium subjected to intermittent anaerobiosis during long
propagation in vitro. J. Exp. Med., 97: 525-552.
Ham, A. W. 1930 A histological study of the
early phases of bone repair. J. Bone and Joint
Surgery, 12: 827-844.
Rodbard, S. 1958 A method for the induction
of intravascular structures. Circulation, 18:
771-772.
1962 Effect of mechanical forces on
structure of vascular system. Subsect
to 6, pp. 41-51, Chapter 2. Blood Vesst
Lymphatics. ed. David I. Abramson. Ac
Press, N. Y.
Williams, R. G., and B. Roberts 1950 ii cmproved tantalum chamber for prolongc
,'croscopic study of living cells in m
Anat. Rec., 107: 359-374.
Williams, R. G. 1957 A study of bone
from autografts of marrow in rabbits.
129: 187-210.
1961 A steel chamber for long
term microscopic study of living tiss e 1
mammals. Ibid., 139: 37-44.
1962 Comparison of living autogell,sus
and homogenous grafts of cancellous 5 I
heterotopically placed in rabbits. Ibid., I
93-106.
9
-
PLATES
PLATE 1
EXPLANATION
O F FIGURES
In all figures masked round number at the left is the figure number
and those at the right refer to the month and day on which the photomicrograph was made.
Figures 2 to 7, all enlarged 14 X, illustrate a n osteoid mass that arose
spontaneously. The tip of the piece was cut off and transplanted as shown
in figure 3. Figures 3 to 7 inclusive illustrate that the transplanted piece
underwent no significant change over a period of about four months and
stimulated no vessel formation or leucocyte infiltration. It is shown at
higher magnification in figure 15. The arrows in figures 4, 5 and 6 point
to a small fragment of osteoid dislodged when the excision was made.
The piece slowly increased in size, changed shape and fused with the
main mass. It is shown at higher magnification after fusion in figure 14.
102
CARTILAGE AND OSTEOID
PLATE 1
Roy G. Williams
103
PLATE 2
EXPLANATION OF FIGURES
8-13
104
Photomicrographs illustrating size changes over about five months
in an osteoid mass that arose in the remains of a cancellous bone
autograft. X 28.
CARTILAGE AND OSTEOID
Roy G. Williams
PLATE 2
105
PLATE 3
EXPLANATION OF FIGURES
14
Higher magnification of the lower corner of the main osteoid mass
shown i n figure 6. It shows that the separated fragment, at the
arrow i n figure 4, has changed shape and fused with the main mass.
X 125.
15
Higher magnification of the transplanted piece shown in figures 3-7.
Photo made on 1-3. x 125.
16 Enlargement showing cells near the center of figure 2. Cells were
untreated and piece was in the living animal. Cells i n osteoid appear
black unlike those in growing autografts of cancellous bone as shown
in figures 18 and 19. x 230.
106
17
Enlargement of the notch at the upper margin of the mass in figure 12.
It illustrates the laminated spindle-shaped cells that increased the
osteoid by apposition. The arrow points to a new layer forming, This
method of growth was quite different from that shown in figures 18
and 19. X 230.
18
The edge of a n autograft of cancellous bone - the black line below
center. Attached to the edge is, between the arrows, the peripheral
osteogenic fibrous layer in a dormant state. x 230.
19
The edge of a n autograft of cancellous bone during active osteogenesis. The edge of the graft is the lower black line. It illustrates
how a fibrous layer like that shown in figure 18 changes during
osteogenesis. X 230.
CARTILAGE AND OSTEOID
PLATE 3
Roy G. Williams
107
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