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The influence of function on chondrogenesis at the epiphyseal cartilage of a growing long bone.

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The Influence of Function on Chondrogenesis at the
Epiphyseal Cartilage of a Growing Long Bone'
MURRAY C . MEIKLE
Department of Anatomy, University of Cambridge,
Cambridge C B 2 3DY, England
ABSTRACT
In order to study the behaviour of epiphyseal cartilage in a nonfunctional environment, the third metacarpal bone was transplanted intracerebrally as an isograft between 7-day-old litter-mate rats. Host animals were killed
1,2,3,4,
5 , 6 weeks post-operatively, and the cellular kinetics evaluated by means
of tritiated thymidine autoradiography. The study was also used to compare the
effects of function on chondrogenesis at the epiphyseal cartilage with that previously demonstrated for the condylar cartilage of the mandible (Meikle, '73a).
Transplantation resulted in three major changes in the cartilage; there was a
decreased rate of proliferative activity in the cell columns; the cartilage failed
to maintain a satisfactory increase in transverse diameter; the cells of the perichondrium differentiated into osteoblasts instead of chondroblasts. Autoradiographic and histological findings suggested that the inability of the cartilage to
increase in transverse diameter was related to the decreased rate of proliferative
activity in the cell columns and not to the cessation of perichondrial chondrogenesis. On the basis of these findings two conclusions can be made.
1. Extrinsic mechanical stresses associated with function appear to be necessary for the normal interstitial growth of epiphyseal cartilage during postnatal
development, suggesting that functional activity can influence the rate of cell
proliferation.
2. Functional activity provides the stimulus for the differentiation of perichondrial cells into chondroblasts.
It has been demonstrated by many investigators (Willis, '36; Lacroix, '51 ; Felts,
'59; Chalmers, '65; Noel, '73) that embryonic and post-embryonic long bones transplanted into a non-functional environment
are capable of achieving growth levels
comparable to that of the bone in situ.
Nevertheless, despite general agreement
that the longitudinal growth exhibited by
such transplanted bones is due principally
to chondrogenesis at the epiphyseal plate,
the role of function in growth of epiphyseal
cartilage is poorly understood. The objectives of this investigation therefore were
twofold. First, to compare the growth of
epiphyseal cartilage in functional and nonfunctional environments; second, to compare the influence of function on chondrogenesis at the epiphyseal cartilage with
that previously demonstrated for condylar
cartilage (Meikle, '73a).
ANAT. REC., 182: 387-400.
MATERIALS AND METHODS
Inbred rats of the Buffalo Strain (Simonsen Laboratories, California) were used in
this study. The third metacarpal was
chosen for the experiment because of its
convenient size, Transplantation was always performed between 7-day-old rats of
the same litter, and always into the intracerebral site.
Experimental procedure
Using an aseptic surgical technique the
third metacarpal was dissected free of
the donor forelimb and placed in Ringer's
solution until all the transplants had been
collected. Under ether anaesthesia a midline incision approximately 1 cm. in length
was made in the scalp of the host and the
Received June 11, '74. Accepted Dec. 18, '74.
1 Supported by a Grant (MA-4020) from the Medical
Research Council of Canada.
387
388
MURRAY C. MEIKLE
transplant inserted deep into the left cerebral hemisphere via a V-shaped incision in
the parietal bone. The scalp incision was
then closed with interrupted black silk
sutures.
The host rats were killed at 1, 2, 3, 4, 5
and 6 weeks post-transplantation; there
were five animals in each group. Each rat
received an intraperitoneal injection of
tritiated thymidine two and four hours before death. The dosage was in the order of
1 &i/gm. body weight (New England Nuclear, specific activity 6.7 Ci/mM). Further
information concerning cell differentiation
was obtained from two additional groups
of five animals which received the same
dosage of tritiated thymidine at one and
three weeks post-transplantation, but were
killed 1, 3, 5, 7 and 10 days later. A control was provided for each transplant by
removing a third metacarpal bone from
the host forelimb.
Histological preparation
The host animals were killed with ether
and the decapitated heads together with
one of the forelimbs fixed in 10% formol
saline. After decalcification in 5% formic
acid-5% formaldehyde, the metacarpals
were embedded in paraplast and longitudinal sections cut at 5 I*m.The sections were
mounted on glass slides and dipped in
Kodak NTB-2 emulsion. Exposure was carried out in light-tight slide boxes at 4°C
for four weeks. The slides were developed
in Kodak Dektol developer for three minutes and fixed for five minutes in 30%
sodium thiosulphate solution. Autoradiographs were stained with Harris haematoxylin. Harris haematoxylin and eosin and
alcian blue were used to stain the routine
histological sections.
Measurements and calculation
of results
To obtain a measure of the effect of
transplantation on the proliferative activity
of epiphyseal cartilage, a comparison was
made between the number of labelled cell
columns in each of the experimental and
control groups (fig. 1). To determine the
effect of transplantation on the transverse
diameter of the cartilage, the number of
cell columns per epiphyseal plate was
selected as the most satisfactory means of
1
1
1
0
1
2
3
4
5
6
TRANSPLANTATION INTERVAL (WEEKS)
Fig. 1 A comparison of the mean number
(and range) of labelled cell columns in the
epiphyseal cartilage of the transplanted and control metacarpals. This parameter was selected as
a measure of the effect of transplantation on proliferative activity within the cartilage. 0 . . . 0
transplanted metacarpals, 0
0
control meta-
. .
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8
4
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1
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4
5
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TRANSPLANTATION INTERVAL (WEEKS)
TIME OF
TRANSPLANTATION
Fig. 2 A comparison of the mean number (and
range) of cell columns in the epiphyseal cartilage
of the transplanted and control metacarpals. This
parameter was selected as a measure of the effect
of transplantation on the transverse diameter of
the cartilage. 0 . . . . . 0 transplanted metacarpals, 0
0
control metacarpals.
INFLUENCE OF FUNCTION ON CHONDROGENESIS
quantifying changes in this parameter
(fig. 2). It is emphasized however, that
given the difficulties of selecting suitably
representative sections for analysis and
distinguishing between adjacent cell columns such data is very subjective. Apart
from the 4-6 week experimental groups, in
which disorganization of the cartilage in
some transplants made analysis difficult,
the data was derived from four sections of
each of four transplanted and control metacarpals. Each point on the two graphs represents the mean and range of the four
observations. The approximate curves of
growth were drawn in by eye.
RESULTS
The general morphological features of
the transplants were comparable to the
controls, although there was a time lag in
the appearance of the secondary ossification centre at the epiphysis. As is usual in
the case of transplanted skeletal tissues,
some cell death occurred as a consequence
of the interference to the blood supply and
nutritional requirements of the cells.
During the fist three weeks the number
of actively dividing cell columns within
the epiphyseal cartilage of the transplants,
although reduced, was not strikingly different from that of the controls (fig. 1). What
was striking however, was the different
rate at which cell division was occurring
as indicated by the number of labelled cells
per column. It was usual to see several
labelled cells per column in the controls
(fig, 6), whereas in the transplants, one,
or at most two labelled cells was the rule
(fig. 4).
A significant difference in the arrangement of the cell columns was also apparent, In the transplants the columns were
typically parallel to each other composed
of a single line of cells (figs. 3, 7), an
arrangement also seen in the 5-6 week
control metacarpals (fig. 16) where the
rate of proliferative activity of the columns
had also decreased (fig. 17). In contrast,
the columns of the controls were made up
of broad bands of interdigitating cells, frequently giving rise to two columns of degenerating chondrocytes (fig. 8).
While the rate of cell division clearly decreased as a result of transplantation. the
differentiation of proliferative zone cells
389
into chondroblasts did not appear to be
affected, as shown by the eventual appearance of labelled chondroblast/chondrocytes (fig. 9 ) in the time course studies.
From 4-6 weeks, while the decline in
proliferative activity continued (fig. 1),
there was some variation in the effect of
transplantation on the form and activity
of the cartilage. Some transplants showed
a complete absence of cell division and
loss of structural organization (fig. 18).
Others however remained relatively well
organized with cell division very much in
evidence (fig. 19).
Using the number of cell columns per
cartilage plate as a measure of the changes
in the transverse diameter or circumference of the cartilage during transplantation and growth, it was obvious that the
transplants showed a very small increase
in this parameter (fig. 2). The differences
even after two weeks of transplantation
are obvious from a comparison of figures
3 and 5, both of which are at the same
magnification. Normally the edges of the
epiphyseal cartilage plate, in addition to
being continuous with the cartilage of the
rest of the epiphysis gradually blend into
the tissues comprising the perichondrium
(figs. 5, 10). In the transplants however
the edges of the cartilage terminated
abruptly (figs. 3 , 11). While autoradiography revealed the presence of many
labelled progenitor cells in the perichondrial zone (fig. 14), it was apparent that
instead of differentiating into chondroblasts as normal, these cells were differentiating into osteoblasts and laying down
bone at the edge of the cartilage, as shown
by the incorporation of labelled osteoblasts/osteocytes into the bone matrix
(figs. 14, 15). Figure 12 illustrates the
initial stage of perichondrial osteogenesis.
The quite distinct line of demarcation that
was usually found to exist between the
epiphyseal cartilage and the perichondrial
bone is shown in figure 13.
DISCUSSION
The way in which epiphyseal cartilage
increases in transverse diameter during
growth has been the subject of frequent
debate since the time of Ranvier (1873)
and Schafer (1878). The point at issue is
whether the increase results entirely from
390
MURRAY C. MEIKLE
the interstitial growth of existing cell
columns, or whether the differentiation
of perichondrial cells into chondroblasts
makes any significant contribution.
In the present study the inability of
transplanted epiphyseal cartilage to increase in transverse diameter was due to
the decreased rate of proliferative activity
within the cell columns and not to the
cessation of perichondrial chondrogenesis.
Since transplanted long bones are capable
of achieving longitudinal growth levels
comparable to those of the bone in situ,
the cell columns continued to divide at
rates that were sufficient to account for
length increments approaching normal
levels, but insufficient to provide the additional columns necessary to maintain a
normal increase in transverse diameter.
One must agree therefore with the conclusions of Riga1 ('62) and Hert ('72) that
epiphyseal cartilage increases in transverse
diameter by interstitial growth. The experimental evidence also suggests that for
normal interstitial growth of epiphyseal
cartilage to occur during post-natal development, functional activity is required. While
perichondrial chondrogenesis is obviously
important during the growth and remodelling of the epiphysis of a long bone, its
role in the growth of epiphyseal cartilage
itself can only be a minor one. Autoradiographic time course studies of periosteal
osteogenesis and chondrogenesis (Owen,
'70; Blackwood, '66) have shown that
osteoblasts and chondroblasts, unlike the
progenitor cells from which they differentiate, do not divide so that it is highly
improbable that chondroblasts from the
perichondrium make a direct contribution
to the cell columns at the extremities of
the plate.
In any transplantation experiment such
as this, given that the ages of the donor
and host are identical, the most obvious
environmental difference between the experimental and control bones is the absence of functional activity. While one can
suggest that functional activity provides
the stimulus for perichondrial chondrogenesis with relative confidence, caution
must be manifest in interpreting the decreased rate of proliferative activity in the
epiphyseal cartilage of the transplants as
being due to the absence of functional
stimuli. Clearly, re-establishment of the
blood supply to a transplant is important if
retardation of growth is to be avoided, and
although the low metabolic demands of
cartilage enable i t to survive transplantation better than other skeletal tissues, interference to the vasculature affected the
vitality of some chondrocytes in a few
transplants. Nevertheless, Felts ('61 ) in a
comprehensive review of transplantation
was of the opinion that intracerebral
transplants appeared to be revascularized
better, and perhaps more rapidly than
those in more common sites. Moreover,
once established, the level of vasculature
was sufficient to provide not only for the
appearance of the secondary ossification
centre, but also to sustain both periosteal
and endochondral osteogenesis. Since osteogenesis requires a higher level of vascularity than chondrogenesis it seems
reasonable to attribute the decreased rate
of proliferative activity in transplanted
epiphyseal cartilage to the absence of functional activity rather than to a defective
blood supply.
The ability of the perichondrium of a
transplanted long bone to switch from
chondrogensis to osteogenesis is similar to
the response shown by the proliferative
zone of the mandibular condyle to transplantation. Autoradiographic studies with
tritiated thymidine (Dale et al., '63; Blackwood, '66), have shown that the progenitor
cells of the proliferative zone differentiate
into the chondroblasts of condylar cartilage. However, when the mandibular joint
of the rat was transplanted intracerebrally
into a nonfunctional environment (Meikle,
'73a), it was found that the progenitor
cells differentiated into osteoblasts. Transplantation experiments, therefore, suggest
that the progenitor cells of perichondrium
and proliferative zone are multipotential
and can form cartilage or bone depending
upon the environmental circumstances.
Such behaviour is not unexpected, since
both are equivalent to the cellular layer of
the periosteum, and it has been demonstrated by several investigators that under
certain conditions periosteum can form
cartilage (Ham, '30; Fell, '33; Glucksmann,
'42; Felts, '61).
Hall ('70) in a review of cellular differentiation in skeletal tissues has pointed
INFLUENCE OF FUNCTION ON CHONDROGENESIS
out that the common initiating factors in
evoking either osteogenesis or chondrogenesis, are the degree of vascularity of the
tissues and the presence or absence of
mechanical stresses. It has been shown in
vitro that hypoxia enhances osteogenesis
and anoxia chondrogenesis (Bassett, '64;
Goldhaber, ' 6 3 ) , and Hall suggests that
mechanical stresses by producing ischaemia, may induce osteogenic cells to become
chondrogenic. Meikle ('73a,b) concluded
that the stimulus for chondrogenesis in the
mandibular condyle was provided by extrinsic mechanical stimuli arising from
the functional activity of the mandibular
joint. In the case of a long bone, it is probable that mechanical stimuli arising from
functional activity and transmitted to the
periosteum via the musculature, are also
responsible for the differentiation of progenitor cells from the perichondrium into
chondroblasts.
ACKNOWLEDGMENTS
The author wishes to express his gratitude to Mrs. Barbara Tait, Department of
Orthodontics, Faculty of Dentistry, University of British Columbia who prepared
the histological material, and to Dr. C. W.
M. Pratt, Department of Anatomy, University of Cambridge, for his invaluable
comments during the preparation of the
manuscript.
LITERATURE CITED
Basset, C. A. L. 1964 Environmental and cellular factors regulating osteogenesis. In: Bone
Biodynamics. H. M. Frost, ed. Little, Brown &
Co., Boston, pp. 233-244.
Blackwood, H. J. J. 1966 Growth of the mandibular condyle of the rat studied with tritiated
thymidine. Archs. Oral Biol., 11: 403-500.
Chalmers, J. 1965 A study of some of the factors controlling growth of transplanted skeletal
tissue. In: Calcified Tissues. L. J. Richelle and
M. J. Dallemagne, eds. Universite de Li&ge,
pp. 177-184.
39 1
Dale, J., A. Hunt, G. Pudy and D. Wagener 1963
A n autoradiographic study of the temporomandibular joint. J. Can. Dent. Ass., 29: 27-28.
Fell, H. B. 1933 Chondrogenesis in cultures of
endosteum. Proc. Roy. SOC.B., 112: 417427.
Felts, W.J. L. 1959 Transplantation studies of
factors in skeletal organogenesis. I. The subcutaneously implanted immature long bone of
the rat. Am. J. Phys. Anthrop., 71: 201-215.
1961 I n vivo implantation as a technique i n skeletal biology. Int. Rev. Cytol., 12:
243-302.
Glucksmann, A. 1942 The role of mechanical
stresses on bone formation in &To. J. Anat.,
76: 231-236.
Goldhaber, P. 1963 Some chemical factors influencing bone resorption in tissue culture.
Publs. Am. Assoc. Adv. Sci., 75: 609-636.
Hall, B. K. 1970 Cellular differentiation in
skeletal tissue. Biol. Rev. Camb. Phil. SOC.,45:
455484.
Ham, A. W. 1930 An histological study of the
early stages of bone repair. J. Bone Jt. Surg.,
12: 827-844.
Hert, J. 1972 Growth of the epiphyseal plate
in circumference. Acta. Anat., 82: 420-436.
Lacroix, P. 1951 The Organization of Bones.
Translated by S. Gilder), Blackiston Co.,
Philadelphia.
Meikle, M. C. 1973a I n vivo transplantation of
the mandibular joint of the rat: an autoradiographic investigation into cellular changes a t
the condyle. Archs. Oral Biol., 18: 1011-1020.
1973b The role of the condyle in the
postnatal growth of the mandible. Am. J. Orthodont., 64: 50-62.
Noel, J. 1973 The control of growth in transplanted mammalian cartilage. J. Embryol. Exp.
Morph., 29: 53-64.
Owen, M. 1970 The origin of bone cells. Int.
Rev. Cytol., 29: 213-238.
Ranvier, L. 1873 Quelques faits relatifs au development du tissue osseux. C. R. Acad. Sci.
(Paris), 77: 1105-1109.
Rigal, R. W. 1962 The use of tritiated thymidine in studies of chondrogenesis. In: Radioisotopes and Bone. P. Lacroix and A. Budy, eds.
Blackwell, Oxford, pp. 197-219.
Schafer, E. A. 1878 Notes on the structure and
development of osseus tissue. Quart. J. Micr.
Sci., 18: 132-141.
Willis, R. A. 1936 The growth of embryo bones
transplanted whole in the rat's brain. Proc. R.
SOC. (London) B., 120: 496-498.
PLATE 1
Two-week metacarpal transplant. Note how the edges of the cartilage terminate
abruptly in comparison to the control (fig. 5). The boxed area corresponds to the autoradiograph shown in figure 4. x 96.
Autoradiograph of a 2-week metacarpal transplant. Although many of the cell columns
exhibit proliferative activity, only one or at most two labelled cells (arrows) are
present in each column. x 262.
Two-week control metacarpal, ( 3 weeks of age). The edges of the cartilage gradually
blend with the tissues comprising the perichondrium, see figure 10 also. The boxed area
corresponds to the autoradiograph shown in figure 6. x 96.
Autoradiograph, 2-week control metacarpal ( 3 weeks of age), illustrating several labelled
cells i n each of the proliferating columns. Arrows indicate columns in which five or
more labelled cells are present. x 262.
3
4
5
6
EXPLANATION OF FIGURES
393
PLATE 2
EXPLANATION OF FIGURES
7
Two-week metacarpal transplant showing the typical arrangement of
the cell columns. Each column consists of a single line of cells. x 390.
8
Two-week control metacarpal ( 3 weeks of age), showing typical
arrangement of the cells columns. x 390.
9
Autoradiograph, transplanted metacarpal (1 week and 1 day).
H3-thymidine administered to host one week after transplantation
and killed 24 hours later. The increased number of labelled cells and
the diminution of the label indicates that chondrogenesis continued
following transplantation.
10 Perichondrial zone 2-week control metacarpal ( 3 weeks of age).
X 250.
11
394
Perichondrial zone 2-week metacarpal transplant. The epiphyseal
cartilage plate ( E C ) terminates abruptly and is lined by progenitor
cells (arrows). Disorganization of the cell columns is evident. Fibrous
periosteum (FP). x 250.
INFLUENCE OF FUNCTION O N CHONDROGENESIS
Murray C. Meikle
PLATE 2
395
PLATE 3
EXPLANATION O F FIGURES
12 The onset of perichondrial osteogenesis (arrows) in a transplanted
metacarpal (1 week and 5 days). x 250.
13 Illustrates the perichondrial bone (PB) abutting the epiphyseal
cartilage (EC) in a 3-week metacarpal transplant. x 375.
14
Autoradiograph showing labelled progenitor cells (arrows) in the
perichondrial zone (PZ) and the perichondrial bone (PB) abutting
the epiphyseal cartilage ( E C ) of a transplanted metacarpal (1 week
and 1 day). H3-thymidne administered to host 1 week after transplantation and killed 24 hours later. X 540.
15 Autoradiograph illustrating the presence of labelled osteoblasts
(arrows) i n an area of perichondrial osteogenesis, transplanted metacarpal (1 week and 3 days). H3-thymidine administered to host one
week after transplantation and killed three days later. x 540.
396
INFLUENCE OF FUNCTION ON CHONDROGENESIS
Murray C. Meikle
PLATE 3
397
w
W
co
PLATE 4
Autoradiograph from a 5-week control metacarpal. Relatively few columns are undergoing proliferative activity judging by the number of labelled cells (arrows) present.
x 262.
17
19
Six-week metacarpal transplant with a well organized cartilage plate, i n which several
columns continue to show proliferative activity. x 225.
18 Five-week metacarpal transplant illustrating the loss of structural orientation that
occurred in some transplants. Arrows indicate perichondrial osteogenesis. x 250.
Five-week control metacarpal ( 6 weeks of age). The columns are composed of a single
line of cells, the arrangement seen in the early transplants (fig. 7 ) . x 275.
16
EXPLANATION O F FIGURES
W
co
co
INFLUENCE OF FUNCTION ON CHONDROGENESIS
Murray C. Meikle
PLATE 4
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