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Copyright 1986 by
Th~
Journal 0/ BOJl~ and Joint
Surg~ry. Incorporal~d
Morphometric Analysis of Chondrocyte Hypertrophy*t+
BY JOSEPH A. BUCKWALTER, M.D.§, DONALD MOWER§, ROBIN UNGAR, B.S.§, JANICE SCHAEFFER§, AND
BARRY GINSBERG, M.D., PH.D.§, IOWA CITY, IOWA
. From the Department of Orthopaedics. Veterans Administration Medical Center and the University of Iowa.
and the Department of Internal Medicine. University of Iowa. Iowa City
ABSTRACT: In the hypertrophic zone of the cartilaginous growth plate, chondrocytes enlarge, assume a
more spherical shape, and form a population of cells
called the hypertrophic chondrocytes. The mechanisms
that are involved in the formation of hypertrophic chondrocytes are poorly understood. Cell hypertrophy usually refers to an increase in cell size and volume
associated with an increase in organelles. In this study,
we sought to determine whether the formation of hypertrophic chondrocytes represents true cell hypertrophy associated with an increase in organelles or whether
it is due to swelling and fluid accumulation. Morphometric analyses of electron micrographs were carried
out to determine changes in cell number, cell volume,
cell organelle volumes, and matrix volumes in the reserve
zone, upper proliferative zone, lower proliferative zone,
upper hypertrophic zone, and lower hypertrophic zone.
Between the upper proliferative zone and the lower hypertrophic zone, the cells increased their mean volume
more than 500 per cent. As they enlarged, their matrices
altered; territorial matrix volume increased as its collagen content decreased, and interterritorial matrix volume decreased as its collagen content increased between
the lower proliferative zone and the lower hypertrophic
zone. Between the upper proliferative zone and the lower
hypertrophic zone, the absolute volume per cell of endoplasmic reticulum, Golgi membranes, and mitochondria increased 126 per cent, while the volume of cytoplasm and nucleoplasm increased 779 per cent, apparently by accumulation of water. Cell organelles of the
lower hypertrophic zone did not show the changes that
are associated with cell injury or death. Thus, the synthesis of organelles contributed to chondrocyte enlargement, but the primary mechanism of cell enlargement
was cytoplasmic and nuclear swelling.
CLINICAL RELEVANCE: During formation of the
skeleton, fracture-healing, and bone growth, enlargement of the chondrocytes contributes to the growth of
cartilage and is closely associated with mineralization of
* This article was accepted for publication prior to July I, 1985. No
conflict-of-interest statement was requested from the authors.
t Read in part at the Annual Meeting of the Orthopaedic Research
Society, Las Vegas, Nevada, January 22, 1985.
t Supported by National Institutes of Health Grant AM 30944-01.
§ University of Iowa, Iowa City, Iowa 52242. Please address reprint
requests to Dr. Buckwalter.
VOL. 68-A, NO.2, FEBRUARY 1986
the matrix and vascular invasion. This study helped to
define the mechanism of the chondrocyte enlargement
that occurs in the hypertrophic zone of the cartilaginous
growth plate.
Chondrocyte hypertrophy, the process by which chondrocytes enlarge and assume a more spherical shape, occurs
during formation of the skeleton, fracture-healing, and longitudinal bone growth 3.4.13.ls.2o.24.26.39.47.s3.59.64. Matrix mineralization and vascular invasion usually follow chondrocyte
hypertrophy, and several authors 7.9.s2 have suggested that
chondrocyte hypertrophy not only contributes to cartilage
growth but also prepares the interterritorial matrix for mineralization or induces mineralization. Despite the apparent
importance of chondrocyte hypertrophy for cartilage growth
and for endochondral ossification, this process remains
poorly understood.
Cells enlarge by increasing their volume of organelles
and cytoplasm; by accumulating lipid, protein, glycogen,
complex carbohydrates, or other material; or by swelling.
Generally, cell hypertrophy refers to enlargement of cells
resulting from an increase in dry mass produced by an increase in the volume or number of organelles43 .44 . Increased
functional demands or hormonal stimulation causes hypertrophy. Examples include phenobarbital-induced synthesis
of endoplasmic reticulum in liver cells; hormonally induced
synthesis of proteins by uterine smooth-muscle cells during
pregnancy; and the increases in endoplasmic reticulum, ribosomes, and myofilaments in cardiac and skeletal-muscle
cells stimulated by increased work43 .44 .
Is the process of chondrocyte enlargement hypertrophy? In 1900, Retter described chondrocyte enlargement
and applied the term hypertrophy to this process S3 .
Others21.26.33.36.s3 adopted Retter's terminology and offered
various explanations for the cell enlargement, including
degeneration 21 .s3 ; vacuolization of the cytoplasm with accumulation of fat and glycogen36; maturation, hypertrophy,
and hydration47 ; and fluid accumulation4.22.48.
Intracellular accumulation of fluid due to cell injury or
degeneration provides a possible explanation of chondrocyte
enlargement. Oxygen tension drops significantly from the
proliferative to the hypertrophic zone8, and experimentally
even temporary hypoxia rapidly leads to cellular edema as
the cell accumulates sodium followed by an iso-osmotic gain
of water·24.44.SS.S6.S8. Alternatively, chondrocyte enlargement
243
244
J. A. BUCKWALTER ET AL.
may be a form of hypertrophy. Several studies have suggested that enlarging chondrocytes are not injured or degenerating. The cells appear to be metabolically active, their
membranes remain intact, and they may have a greater absolute volume of organelles per cell than do other growthplate cells9.I 2.25.26.29-32 .
The purpose of our study was to help to explain chondrocyte enlargement by answering the following questions.
Do growth-plate chondrocytes enlarge by the accumulation
of cell products or metabolites, by hypertrophy, by swelling,
or by a combination of these mechanisms? Do the organelles
of enlarging chondrocytes show the changes associated with
cell injury or degeneration? Do enlarging chondrocytes
modify the volume or structure of their extracellular matrix?
components (proximal epiphysis, proximal physis, proximal
metaphysis, marrow, distal metaphysis, distal growth plate,
and distal epiphysis).
The methods for conventional fixation and fixation with
ruthenium hexamine trichloride were recently described in
detail '3 . For electron microscopic study, we immersed the
eight right tibial physes in fixation medium and divided the
central portions into four longitudinally oriented rectangular
blocks. We studied only the central region of the growth
plates, since it produces longitudinal growth while the more
peripheral regions may contribute to circumferential
growth 23 . Four growth plates (sixteen blocks) were fixed
using conventional methods and four growth plates (sixteen
blocks) were fixed using ruthenium hexamine trichloride.
Forty to sixty-nanometer-thick sections were cut from each
block and transmission electron micrographs were made
with a Hitachi 600-H electron microscope.
Methods
Animal Model and Tissue-Processing
Eight fifteen-day-old C57 male mice (Harlan Sprague
Dawley, Indianapolis, Indiana) were anesthetized with ether
and killed by cervical dislocation. The tibiae were removed;
the left tibia was used for light microscopic study and the
right tibia, for electron microscopic study.
For light microscopic study, eight bones were immersed in 10 per cent phosphate buffered formalin (pH 7.3),
decalcified in Cal-Ex (Fisher Scientific) containing 10 per
cent formalin, dehydrated in alcohol, and embedded. Fivemicrometer-thick longitudinal sections were mounted on
glass slides and stained with hematoxylin and eosin . From
each bone, we cut six to ten sections that contained all tissue
LEVEL I
LEVEL II
LEVEL '"
Morphometric Measurement and
Tabulation of Measurements
Using a morphometric model of the tibia and proximal
physis (Fig. I), we performed the following measurements.
Level I - the whole bone: Bone volumes were determined by placing the tibiae in a calibrated glass cylinder
containing fixative and measuring the volume displacement
using a microliter syringe. Length was measured with a
micrometer.
Level 2 - the bone-component tissues: Measurements
of bone-component tissues were made from the images of
Tibia
Proximal
Epiphysis
Proximal
Physis
I
Reserve Zone
Matrix
I
Pericellular &
I
Distal
Epiphysis
I
Upper
Hypertrophic
Zone
Lower
Hypertrophic
Zone
I
I
I
I
Distal
Physis
I
Lower
Proliferative
Zone
I
LEVEL IV
Marrow
I
Upper
Proliferative
Zone
I
I
Diaphysis &
Metaphysis
I
Cell
I
I
Interterritorial
Territorial
LEVEL V
Lipid
EndoplasmiC
Reticulum
Lucent
Space
I
I
Normal
Dilated
Membrane
Bound
Non-membrane
Bound
Vacuoles
~
Large
Small
FIG . I
Morphometric model of the tibia and physis. We examined only the proximal tibial physis at levels 3, 4, and 5. We examined all five growth-plate
zones at levels 4 and 5.
THE JOURNAL OF BONE AND JOINT SURGERY
MORPHOMETRIC ANALYSIS OF CHONDROCYTE HYPERTROPHY
six to ten whole bone sections from each tibia, projected
with a Leitz Laborlux-II microscope at a magnification factor of twelve onto a Houston Instrument Hi-Pad interfaced
with an Apple lIE computer. Epiphyses were defined as the
regions extending from the surface of the joint to the top
of the resting zone of the physis. Physes consisted of the
regions between the top of the resting zone and the last
intact transverse septum of the hypertrophic zone. Diaphyses and metaphyses were defined as the regions between
hypertrophic zones, excluding the hematopoietic and fatty
marrow. Component tissue areas were determined by planimetry using an image-analysis program (R & M Biometrics; Nashville, Tennessee). The mean value for all sections
of a given bone was accepted as the value for that bone.
Level 3 - growth-plate zones: Growth-plate-zone volumes were determined by projecting the light microscopic
sections of growth plate at a magnification factor of 100
onto the digitizing pad. We defined the growth-plate zones
as follows l3 • The reserve zone included the region containing single cells or small clusters of cells between the epiphyseal bone and the top of the cell columns. The proliferative
zone consisted of the region beginning at the top of the
columns of flattened cells and extending to the point where
the density of cell cytoplasm decreased and the cells began
to enlarge. The hypertrophic zone included the region from
the bottom of the proliferative zone to the last intact transverse septum. The proliferative and hypertrophic zones were
divided in half to produce the upper and lower halves of
each zone. The planimetry program was used to determine
the portion of the proximal growth plate that was contributed
by each zone and the mean values for all sections of a given
bone were accepted as the values for that bone.
Level 4 - cells and matrices of growth-plate zones:
We analyzed one section from each block. Seven to ten
electron micrographs were made of each zone of each block
using standardized stratified sampling of zones 45 .46 . In each
micrograph we tabulated the number of cells per unit of
area. Then a 100-point lattice grid was superimposed on the
electron micrographic images and the number of points falling on cells, pericellular and territorial matrix, and interterritorial matrix was recordedI6.17,40.41. Since the volume of
pericellular matrix was very small, we combined the pericellular and territorial matrices. Within the matrices, we
used point-counting to determine the volume fractions of
collagen, ground substance, and matrix vesicles.
Level 5 - cell organelles: For study of the cell organelles indicated in Figure 1, the microscope screens were
positioned on the first five cells encountered within each
zone and micrographs were made at a magnification factor
of 10,000. We grouped membrane-bound structures into
four size classes: those less than 100 nanometers in diameter
were termed vesicles, those less than 400 nanometers in
diameter were considered small vacuoles, those less than
1100 nanometers in diameter were called large vacuoles,
and those larger than 1100 nanometers were termed membrane-bound spaces. The endoplasmic reticulum was considered to be dilated if it exceeded fifteen nanometers in
VOL. 68-A, NO.2. FEBRUARY 1986
245
width. The cytoplasm was divided into electron-dense areas
of cytoplasmic ground substance and cytoplasmic lucent
spaces that were either membrane-bound or non-membranebound. A 100-point lattice grid was superimposed on the
electron micrographs and point-counting was used to determine organelle volume densities.
Determination of Volume Density and Numerical Density
Volume Density
The volume density, Vv - that is, the relative volume
of a component or class of components contained within a
unit of volume - can be estimated from sections by measuring the relative areas of the components, AA 60.61. To
measure the AA of component tissues within the bone and
the AA of growth-plate zones within the growth plate, we
used planimetry. To measure the AA of cells, organelles,
matrices, collagen, ground substance, and matrix vesicles,
we used point-counting.
Numerical Density
We calculated the relative number of cells contained
within a unit of volume 60.62 , N" by two methods: the WeibelGomez method 60 ·61 and the Hilliard method as presented by
Williams 63 .
The Weibel-Gomez method estimates N v from the following formula:
where Nv = numerical density, NA = the number of profiles
per unit area, Vv = the volume density, beta = the shape
coefficient (the dimensionless relationship between particle
volume and mean cross-sectional area), and K = the size
distribution coefficient. Based on a study of growth-plate
cell-profile orientations l3 , we assumed that the probability
of a cell being cut by a longitudinal section of the growth
plate was the same as that of a sphere randomly arranged
in the transverse plane, and we used the shape coefficient
for spheres, beta = 1.38. The size distribution coefficient,
K, was determined for each zone using cell dimensions 13 to
extrapolate from the plot given by Weibel 60 •
The Hilliard method63 estimates Nv from the following
formula: N v = NA/D, where 0 = the mean tangent diameter
of the cells, which was determined by measurement of the
cells of each zone l3 •
Statistical Analysis
Values were expressed as the mean and standard deviation. We performed one-way analysis of variance followed by Tukey's multiple-comparison p'rocedure to
compare values from different growth-plate zones and from
conventionally fixed and ruthenium hexamine trichloridefixed samples50 . We accepted p < 0.01 as indicating a significant difference between mean values.
Results
The proximal tibial growth plate contributed 4.8 ±
246
1. A. BUCKWALTER ET AL.
TABLE I
DIFFERENCES IN CELL NUMERICAL DENSITY AMONG GROWTH-PLATE ZONES*
(IN NUMBER OF CELLS PER MILLILITER X 10")
Conventional Fixation
(16 Blocks)
Growth-Plate Zone
Reserve
Upper proliferative
Lower proliferative
Upper hypenrophic
Lower hypenrophic
Weibel-Gomez
4.21
[ 4.89
t5.43
2.69
1.19
[
± 0.62
±
±
±
±
0.90
1.37
0.83
0.38
Ruthenium Fixation
(16 Blocks)
Hilliard
3.37
l4.73
·4.31
2.14
1.08
* Bars connect means that were not significantly different among zones.
± 0.32
± 0.67
± 0.72
± 0.39
::!:
0.21
Weibel-Gomez
4.84
5.95
t6.59
2.93
1.54
±
±
±
±
±
0.93
0.88
1.40
0.64
0.40
Hilliard
4.39
[ 5.85
t5.43
2.82
1.69
± 0.65
± 0.71
± 1.01
± 0.42
::!:
0.27
All other differences in means among zones were significant (p < 0.01) .
t Mean values that were significantly different between the Weibel-Gomez and the Hilliard methods.
0.2 per cent of bone volume, or 73.0 ± 0.03 X 10- 2
microliters (eight samples). The growth-plate zones made
the following contributions to total growth-plate volume:
reserve zone, 7.8 ± 2.3 per cent; upper and lower proliferative zones, each, 20.3 ± 1.7 per cent; and upper and
lower hypertrophic zones, each, 23.8 ± 1.2 per cent (eight
samples) .
Differences between the Weibel-Gomez
and Hilliard Methods
Since growth-plate cells vary in size and shape within
and among zones, and since the cells have a high degree
of orientation in some zones 13, determining cell numerical
density presented a difficult problem. The Weibel-Gomez
method60 ·61 is valid only for isotropic structures of known
shape. To use this method, we assumed that the cells were
randomly distributed spheres in the planes perpendicular to
the long axis of the bone l3 . Furthermore, the size distribution
coefficient, K, was difficult to determine and small changes
in its value caused large changes in numerical density. The
Hilliard method63 assumes that the cells have spherical or
nearly spherical shapes, and it depends on the mean tangent
diameter of the cells, D, which was difficult to determine
reliably. The recently described dissector method of determining numerical density29.sl does not require assumptions
concerning cell shape and size and can be used in anisotropic
tissues; however, it requires precise measurement of section
thickness.
The Weibel-Gomez method did not demonstrate a difference in cell N v between the reserve zone and the upper
proliferative zone in conventionally fixed samples, and in
conventionally fixed and ruthenium hexamine-trichloride
fixed samples the Weibel-Gomez method produced higher
cell N v values in the lower proliferative zone than did the
Hilliard method (Table I). We estimated cell volumes using
formulae for solids that resemble the cell shapes and compared these estimates of cell volume with the estimates
produced by dividing cell Vv by cell N v (Table II). Cell
volumes calculated from the formulae for solids decreased
from the reserve zone to the lower proliferative zone and
then progressively increased from the lower proliferative
zone to the lower hypertrophic zone, which was consistent
with the appearance of the cells on transverse and longi-
tudinal sections l3 . Cell volumes determined by the Hilliard
method followed this pattern but the cell volumes calculated
using the Weibel-Gomez method decreased between the
upper proliferative zone and the lower proliferative zone .
Because the Hilliard method was simpler and produced values that fit more closely with the apparent differences in
cell number and volume among zones, the following data
were based on the Hilliard method.
Differences in Cells and Matrices
among Growth-Plate Zones
The upper and lower proliferative zones had the greatest cell density (Table Ill). The reserve zone had a lower
cell density than the upper or lower proliferative zone but
a higher cell density than the upper hypertrophic zone. The
upper hypertrophic zone had more cells per milliliter than
the lower hypertrophic zone.
Samples that had been fixed with ruthenium hexamine
trichloride had greater cell N v in all zones (Table Ill) . This
may have occurred because cells fixed with ruthenium hexamine trichloride have different profiles l3 and size and because ruthenium hexamine trichloride increases the contrast
between the cells and the matrix, making it easier to identify
the cells. Despite these differences, fixation with ruthenium
hexamine trichloride and conventional fixation produced the
same pattern of changes in cell N v among zones.
With conventional fixation, cell volume per milliliter
was the same in the reserve, upper proliferative, and lower
proliferative zones (Table III and Fig. 2). Cell volume per
zone increased significantly in the upper hypertrophic zone
and again in the lower hypertrophic zone. In tissue that had
been fixed with ruthenium hexamine trichloride, cell-volume density was higher for every zone than in conventionally fixed tissue and, unlike the results in conventionally
fixed tissue, showed an increase in cell-volume fraction
between the resting zone and the upper proliferative zone.
The cells changed their average volume between zones
(Table Ill). From the reserve zone to the upper proliferative
zone, the average cell volume decreased 21 per cent for
conventionally fixed tissue and 7 per cent for tissue fixed
with ruthenium hexamine trichloride. Cell volume increased
slightly between the upper proliferative zone and the lower
proliferative zone and then increased greatly between the
THE JOURNAL OF BONE AND JOINT SURGERY
247
MORPHOMETRIC ANALYSIS OF CHONDROCYTE HYPERTROPHY
TABLE"
DIFFERENCES IN CELL VOLUME (IN CUBIC MICROMETERS) AMONG GROWTH-PLATE ZoNES
Cell-Volume Fraction (m/lm/)/
Cell Numerical Density (No.lm/)
Calculated from Cell
Axial Dimensionst
Growth-Plate
Zone
Reserve
Upper proliferative
Lower proliferative
Upper hypertrophic
Lower hypertrophic
Assumed Shape
Disk
Disk
Disk
Disk
Rectangular
parallepiped
Rectangular
parallepiped
Formula for
Volume*
Conventional
Fixation
Ruthenium
Fixation
m't
m't
m't
abc
816
576
726
1883
2910
abc
4417
7Trt
Conventional Fixation
Ruthenium Fixation
Weibel-Gomez
Hilliard
Weibel-Gomez
Hilliard
690
610
774
1607
2640
610
585
512
1253
763
605
645
1575
680
686
656
1952
749
697
796
2028
4226
3513
3870
4080
3716
*r
= radius and t = height.
t Cell axial measurements were performed as part of another study 13.
lower proliferative zone and the upper hypertrophic zone:
144 per cent for conventionally fixed cells and 155 per cent
for those fixed with ruthenium hexamine trichloride. The
mean cell volumes increased again between the upper hypertrophic zone and the lower hypertrophic zone: 146 per
cent for conventionally fixed cells and 83 per cent for cells
fixed with ruthenium hexamine trichloride. Between the
upper proliferative zone and the lower hypertrophic zone,
the mean cell volume increased 540 per cent for conventionally fixed cells and 432 per cent for those fixed with
ruthenium hexamine trichloride.
The total matrix volume per milliliter necessarily decreased as cell volume increased (Table III, Fig. 2). Less
predictably, the matrix volume per cell increased as the cell
volume increased. In conventionally fixed tissue, the matrix
volume per cell increased from 1500 cubic micrometers in
the upper proliferative zone to 5370 cubic micrometers in
the lower hypertrophic zone, and in tissue fixed with ruthenium hexamine trichloride it increased from 1010 cubic
micrometers in the upper proliferative zone to 2190 cubic
micrometers in the lower hypertrophic zone. We also found
that the volume of the territorial matrix increased as that of
the interterritorial matrix decreased. With conventional fixation, the volume of the territorial matrix increased from 4
per cent of the reserve zone to 27 per cent of the lower
hypertrophic zone, and with fixation with ruthenium hex-
Conventional Fixation
100
75
50
25
o
RHT Fixation
100
•
Interterritorial Matrix
El
Pericellular and
Territorial Matrices
~ Cells
75
50
25
o
RZ
UPZ
lPZ
UHZ
lHZ
FIG. 2
Histograms illustrating the changes in the relative volumes of cells. pericellular and territorial matrix, and interterritorial matrix among zones. From
the reserve zone to the lower hypertrophic zone. cell and pericellular matrix volume and the volume of the territorial matrix progressively increased at
the expense of the volume of the interterritorial matrix. In all zones, relative cell volume was greater in tissue that had been fixed with ruthenium
hexamine trichloride. However, the degree of difference between tissue fixed with ruthenium hexamine trichloride and conventionally fixed tissue varied
among zones: the differences were greater in the upper and lower hypertrophic zones than in the other zones. RZ = reserve zone, UPZ = upper
proliferative zone, LPZ = lower proliferative zone, UHZ = upper hypertrophic zone, and LHZ = lower hypertrophic zone.
VOL. 68·A, NO.2. FEBRUARY 1986
248
J. A . BUCKWALTER ET AL.
FIG.
3
THE JOURNAL OF BONE AND JOINT SURGERY
MORPHOMETRIC ANALYSIS OF CHONDROCYTE HYPERTROPHY
amine trichloride, it increased from 3 per cent of the reserve
zone to 16 per cent of the lower hypertrophic zone. The
volume of the interterritorial matrix followed the opposite
pattern. With conventional fixation, the interterritorial ma-
249
trix formed 70 per cent of the reserve zone and then steadily
decreased to form only 31 per cent of the lower hypertrophic
zone. Growth plates that had been fixed with ruthenium
hexamine trichloride also demonstrated a steady decrease
FIG. 4
Fig. 3: Electron micrographs of cell cytoplasm, showing little or no change in organelle structure among zones but a decrease in cytoplasmic density
and an increase in the cytoplasmic lucent space between the lower proliferative zone and the upper hypertrophic zone and between the upper hypertrophic
zone and the lower hypertrophic zone (x 20,000). A: Reserve zone, conventional fixation. B: Upper proliferative zone, conventional fixation. C:
Lower proliferative zone, conventional fixation. D: Upper hypertrophic zone, conventional fixation. E: Lower hypertrophic zone, conventional fixation.
F: Reserve zone, fixation with ruthenium hexamine trichloride. G: Upper proliferative zone, fixation with ruthenium hexamine trichloride. H: Lower
proliferative zone, fixation with ruthenium hexamine trichloride. I: Upper hypertrophic zone, fixation with ruthenium hexamine trichloride. J: Lower
hypertrophic zone, fixation with ruthenium hexamine trichloride.
Fig. 4: Electron micrographs of cell nuclei, showing the decrease in density of the nucleoplasm between the lower proliferative zone and the upper
hypertrophic zone and between the upper hypertrophic zone and the lower hypertrophic zone. A: Reserve zone, x 8500. B: Upper proliferative zonC',
x 6800. C: Lower prol iferative zone, x 8500. D: Upper hypertrophic zone, x 5100. E: Lower hypertrophic zone, x 5100.
VOL. 68-A, NO.2. FEBRUARY 1986
250
J. A . BUCKWALTER ET AL.
TABLE
DIFFERENCES IN CELLS AND
Cells
Number
Growth-Plate Zone
Volume
Per ml Tissue
N, (X 1001ml)
(x IIJ'IZone)
Per Zone
Per ml Tissue
V, (mUml)
Mean VoI.lCell
(fJ-')
Conventional fixation
(16 blocks)
Reserve
Upper proliferative
Lower proliferative
Upper hypertrophic
Lower hypertrophic
t3 .37
[t4.73
t4.31
t2 . 14
tl.08
±
±
±
±
±
0.32
0.67
0.72
0.39
0.21
1.8
6.9
6.2
3.6
1.8
~ to .26 ± 0.02
to.29
to.28
to.34
to.42
±
±
±
±
0.02
0.01
0.03
0.04
763
605
645
1575
3870
Ruthenium fixation
(16 blocks)
Reserve
Upper proliferative
Lower proliferative
Upper hypertrophic
Lower hypertrophic
t4.39
[t5.85
t5.43
t2.82
tl.69
±
±
±
±
±
0.65
0.71
1.01
0.42
0.27
2.4
8.5
7.9
4.7
2.9
to.33
[ to.41
to.43
to.57
to.63
±
±
±
±
±
0.03
0.02
0.03
0.02
0.01
749
697
796
2028
3716
• Bars connect means that were not significantly different among zones . All other differences in means among zones were significant (p < 0.01) .
t Indicates mean values that were significantly different between conventionally fixed samples and samples fixed with ruthenium hexamine trichloride (p < 0.01) .
in the proportion of interterritorial matrix, from 64 per cent
in the reserve zone to 21 per cent in the lower hypertrophic
zone.
The relative volumes of matrix collagen and ground
substance changed among zones (Table III). The values for
collagen and ground substance may not add up to one because of the volume occupied by matrix vesicles. In the
territorial matrix, the relative volume of collagen steadily
decreased from 55 per cent of the matrix in the reserve zone
to 6 per cent of the matrix in the lower hypertrophic zone.
In the interterritorial matrix, the relative volume of collagen
decreased from 69 per cent of the matrix in the reserve zone
to 53 per cent of the matrix in the lower proliferative zone
and then increased to 61 per cent of the matrix volume in
the lower hypertrophic zone.
Differences in Cell Structure among Growth-Plate Zones
From the upper proliferative zone to the lower hypertrophic zone, nuclear and cytoplasmic density decreased and
cytoplasmic lucent space increased (Figs. 3 and 4). The
most dramatic changes occurred between the lower proliferative zone and the upper hypertrophic zone and between
the upper hypertrophic zone and the lower hypertrophic
zone.
The proportions of the cells occupied by various organelles changed among zones (Table IV). Between the
reserve zone and the proliferative zone, the per cent of the
cell occupied by nucleus, endoplasmic reticulum, Golgi
membranes, and cytoplasmic ground substance increased
and the per cent occupied by glycogen, large vacuoles, lipid,
and cytoplasmic space decreased. Between the upper proliferative zone and the lower proliferative zone, the per cent
of mitochondria and vesicles decreased. The proportion of
the cell occupied by the nucleus, endoplasmic reticulum,
mitochondria, and Golgi membranes then decreased , and
the proportion occupied by vacuoles and cytoplasmic space
increased between the lower proliferative zone and the upper
hypertrophic zone. The cells of the lower hypertrophic zone
had a further decrease in the proportion 'of their volume
occupied by endoplasmic reticulum, glycogen, Golgi membranes, vesicles, and cytoplasmic ground substance and an
increase in the proportion of cytoplasmic space.
These changes gave the cells of each zone distinctive
features. Reserve-zone cells had the maximum proportion
of lipid and glycogen . Proliferative-zone cells had the maximum proportion of nucleus, endoplasmic reticulum, mitochondria , and Golgi membranes and the minimum
proportion of cytoplasmic space. Hypertrophic-zone cells
had the maximum proportion of cytoplasmic space.
We also examined the cells for changes in organelle
structure. With conventional fixation, the proportion of dilated endoplasmic reticulum increased from the reserve zone
to the hypertrophic zone. With fixation with ruthenium hexamine trichloride, the proportion of dilated endoplasmic
reticulum did not increase (Table IV). With both methods
of fixation, ribosomes remained associated with the membranes of the endoplasmic reticulum. Outer mitochondrial
membranes remained intact in all zones and the preservation
of mitochondrial cristae did not differ among zones (Fig.
3).
Although the changes in organelle volumes among
zones were similar in conventionally fixed tissue and tissue
that had been fixed with ruthenium hexamine trichloride, in
tissue that had been fixed with ruthenium hexamine trichloride glycogen was almost absent from the cells, which
increased the volume of cytoplasm, especially in reservezone cells (Table IV). Cells fixed with ruthenium hexamine
trichloride tended to have a lower proportion of endoplasmic
reticulum , mitochondria, Golgi membranes, vesicles, vacuoles, and lysosomes but a higher proportion of cytoplasmic
space and cytoplasmic ground substance. Presumably these
differences occurred because ruthenium hexamine trichloride fixed the cells in a more expanded form.
To clarify the mechanism that is primarily responsible
THE JOURNAL OF BONE AND JOINT SURGERY
251
MORPHOMETRIC ANALYSIS OF CHONDROCYTE HYPERTROPHY
III
MATRICES AMONG GROWTH-PLATE ZoNES'
Matrices
Pericellular and Territorial Matrix
Per ml Tissue
V. (mllml)
0.04
0.08
[to.14
to.17
to.27
±
±
±
±
±
0.005
0.01
0.01
0.005
0.Q2
[ 0.03
[0.05
tO.07
to.12
to.16
±
±
±
±
±
0.002
0.01
0.003
0.01
0.09
Collagen
V. (mllml)
0.55
0.46
0.34
[0.10
0.06
±
±
±
±
±
0.10
0.12
0.15
0.06
0.05
Interterritorial Matrix
Ground
Substance
V. (mllml)
0.44
0.54
0.66
[0.89
0.94
±
±
±
±
±
0.10
0.12
0.15
0.06
0.05
Collagen
V.(mllml)
Per ml Tissue
V. (mllml)
to.70
to.63
to.59
to.49
to.31
±
±
±
±
±
0.Q2
0.03
0.05
0.03
0.02
to.64
to.54
to.50
to.31
to.21
±
±
±
±
±
0.03
0.03
0.03
0.03
0.Q2
0.69
~0.61
[0.53
0.55
0.61
for cell enlargement, we compared the absolute contributions of various cellular components and the increase in cell
volume between the upper proliferative zone and the lower
hypertrophic zone (Table IV and Fig. 5). Since conventional
fixation and fixation with ruthenium hexamine trichloride
produced similar results (Table IV), we describe here only
the data from conventional fixation. Glycogen and lipid
increased by nineteen cubic micrometers per cell. Vesicles,
vacuoles, and lysosomes increased by 122 cubic micrometers per cell and the endoplasmic reticulum, mitochondria,
and Golgi membranes increased by 211 cubic micrometers
per cell. In contrast, nuclear volume increased by 469 cubic
micrometers per cell; cytoplasmic ground substance, by 630
cubic micrometers per cell; and cytoplasmic space, by 1795
± 0.09
±
±
±
±
0.07
0.07
0.07
0.09
Matrix Volume per Cell
Ground
Substance
V.(mllml)
0.31
~0.39
[0.45
0.43
0.38
Pericellular and
Territorial Matrix
Interterritorial
Matrix
(",3)
(",3)
120
170
325
794
2500
2080
1330
1370
2290
2870
70
85
130
425
950
1460
925
920
1100
1240
± 0.08
±
±
±
±
0.07
0.06
0.06
0.09
cubic micrometers per cell. The increase in the cytoplasmic
space presumably reflected increased fluid, and since the
nucleoplasm and the cytoplasmic ground substance decreased in density as they expanded, most of their increased
volume probably also resulted from the accumulation of
fluid. Therefore, we suspect that cells increased their fluid
volume by as much as 2900 cubic micrometers per cell
between the upper proliferative zone and the lower hypertrophic zone.
Discussion
The changes that we found among chondrocytes from
different growth-plate zones agree with the results of other
studies. The changes in cell numerical density and volume
1800
Increases
1200
in
Absolute
Volume
per Cell
(1-1 3 )
Between UPZ
600
& LHZ
o
Glycogen
Lipid
Vesicles Endoplasmic Nucleus
Vacuoles
Reticulum
Lysosomes Mitochondria
Golgi
FIG.
Cytoplasmic Cytoplasmic
Ground
Space
Substance
5
Histogram showing the increases from the upper proliferative zone (UPZ) to the lower hypertrophic zone (LHZ) in the absolute volume per cell of:
glycogen and lipid; vesicles. vacuoles. and lysosomes; endoplasmic reticulum. mitochondria. and Golgi membranes; nucleoplasm; cytoplasmic ground
substance; and cytoplasmic space. Accumulation of fluid presumably caused the increased volume of nucleoplasm. cytoplasmic ground substance. and
cytoplasmic space volume. We determined the values shown from conventionally fixed tissue; however. tissue fixed with ruthenium hexamine trichloride
showed a similar pattern of changes among zones (Table IV).
VOL. 68-A, NO.2, FEBRUARY 1986
252
J. A . BUCKWALTER ET AL.
TABLE
DIFFERENCES IN CELL ORGANELLES
Endoplasmic Reticulum Volume
Growth-Plate Zone
Regular
Nuclei
Conventional fixation
Mean per cent of cell
Reserve
[
16.9 ± U
Upper proliferative
[ t 21.0 ± 1.2
Lower proliferative
20.4 ± 2.0
Upper hypenrophic
[ 13.3 ± 1.0
15.4 ± 2.4
Lower hypenrophic
Mean absolute volume
3
per cell (11 )
Reserve
129
Upper proliferative
127
Lower proliferative
132
Upper hypenrophic
210
Lower hypenrophic
596
Change from upper proliferative
+369%
to lower hypenrophic
Ruthenium fixation
Mean per cent of cell
Reserve
[
,".6 ± 2.9
t24.9 ± 2. 1
Upper proliferative
Lower proliferative
21.7 ± 2.0
Upper hypenrophic
[ 15.6 ± 0.8
Lower hypenrophic
17.7 ± 3. 1
Mean absolute volume
3
per cell (11 )
Reserve
139
173
Upper proliferative
Lower proliferative
173
Upper hypenrophic
316
Lower hypenrophic
658
+280%
Change from upper proliferative
to lower hypenrophic
[
6.5
[ 9.2
t8.7
5.4
2.9
±
±
±
±
±
0 .6
1.2
1.5
1.4
0.4
50
56
56
85
112
+100%
5.6
9.9
tll.3
6.7
3.1
±
±
±
±
±
1.7
0.7 [
2.5
2.0
0.8
42
69
90
136
115
+67%
Dilated
Total
±
±
±
±
±
8.8
14.4
17.2
12.4
6.6
2.3
5.2
t8 .5
t7.0
t3.7
0.3
1.4
0.7
0.9
0.7
18
31
55
110
143
+361%
68
87
110
195
255
+193%
±
±
±
±
±
8.3
14.4
17.5
11.2
4.3
2.7
4.5
t6.2
t4.5
tl.2
2.2
1.7
1.8
0.2
0.7
20
31
49
91
45
+45%
62
100
139
227
160
+60%
Glycogen
t15.5
t4 .8
t5.5
t6.1
1.3
E
±
±
±
±
±
Mitochondria
8.7
5.0
4 .5
5.6
1.1
[ t3.9
t4.5
[ 2.5
[ 1.9
1.5
118
29
35
96
50
+72%
±
±
±
±
±
0 .6
0 .3
0.2
0.3
0.2
30
27
16
30
58
+ 115%
0.03
~ to.02
" ' '±' 0.'
to.2 ± 0.2
tl.O ± 1.1
0.9 ± 0.8
3
0.1
2
20
33
+32.900%
[t2.6 ± 1.0
t3.7 ± 1.0
E
2.4 ± 0.6
1.8 ± 0.8
1.5 ± 0.6
19
26
19
37
56
+ 115%
Golgi
Membranes
Vesicles
~ t3.3 ± 0.5 [t2.2 ± 0 .8
C9 .0
9.0
3.6
1.7
±
±
±
±
t 2.4 ±
3.2
2.5 [ tl.6 ±
0.7
tl.3 ±
0.3
to.7 ±
17
15
10
21
27
+80%
25
54
58
57
66
+22%
[ 't5.4
"
7.7
3.0
0.9
±
±
±
±
±
1.2
LI
3.5
1.2
0.5
0.7
0.5
0.5
0.2
~ to.6 ± 0.2
tl.O
to.5
to.5
to.2
6
38
61
61
33
- 13%
±
±
±
±
0.4
0.4
0.3
0.1
5
7
4
10
7
0
• Bars connect means that were not significantly different among zones. All other differences in means among zones were significant (p < 0.01).
t Indicates means that were significantly different between conventionally fixed samples and samples fixed with ruthenium hexamine trichloride (p < 0.0 I) .
foUowed the pattern demonstrated by Howell et al. 27 in rib
growth plate. The integrity of the cell organelles of the lower
hypertrophic zone and the increase in cell organelle content
with cell enlargement are consistent with previous reports 9 . 25 28.31.32. Nonetheless, interpreting our results requires awareness of the limitations of our methods.
Tissue preparation alters cells, and greater loss or distortion of cell organelles in one zone relative to others could
invalidate our results. The structural integrity of the cell
organelles in all zones argues against a differential loss or
distortion, but it does not eliminate the possibility. Tissue
preparation also may alter cell and matrix volumes . Compared with conventionally fixed tissue, material that had
been fixed with ruthenium hexamine trichloride had an increased cell volume at the expense of matrix volume, and
the degree of difference between tissue prepared with the
two fixation methods depended on the zone. We cannot
determine if this occurred because ruthenium hexamine
trichloride maintained the cells in an expanded form that
they have in vivo or because ruthenium hexamine trichloride
increased cell volume by decreasing matrix volume. Other
methods of fixation 32 may answer this question.
This study could not demonstrate cell function. We
found that the absolute volume of endoplasmic reticulum,
Golgi membranes, and mitochondria per cell increased as
cell volume increased. We assume that this indicates that
the cells synthesized more organelles as they enlarged, but
it might also result from swelling of the organelles. Furthermore, we cannot prove that the organelles of the enlarging cells continued to function, although the integrity
of their membranes and the cell membranes suggests that
even the largest chondrocytes remained metabolically active.
The use of quantitative methods to study a complex
biological structure also has limitations. A number of approaches have been used to determine the mean volume
densities and numerical densities of biological structures
from histological sections9. 18. 19.28.29.35.42.45-47.5 1.60.64 . Many of
these approaches do not compensate for errors caused by
anisotropic structures, and all of them depend on assumptions concerning the relationships between the areas and
numbers of structures determined from two-dimensional
sections and the three-dimensional volumes and numerical
densities of these structures. It is difficult to empirically test
these assumptions or to determine if they apply equally well
to different structures. Thus, uncritical acceptance of the
quantitative results could lead to erroneous conclusions.
Despite these limitations, our morphometric analyses
help to explain the process of chondrocyte enlargement. The
measurements show that chondrocytes from different
growth-plate zones differ in structure and strongly suggest
that they differ in function. Cells of the reserve zone were
THE JOURNAL OF BONE AND JOINT SURGERY
253
MORPHOMETRIC ANALYSIS OF CHONDROCYTE HYPERTROPHY
IV
AMONG GROWTH-PLATE ZoNES·
Cytoplasmic Space
Vacuoles
Small
[
~
tl.4
tl.O
to.8
tl.O
0.5
±
±
±
±
±
0.1
0.2
0.1
0.2
0.1
Large
~[fLO
t2.9
II
6
5
16
19
+217%
~
to.3
to.4
to.4
to.2
0.3
±
±
±
±
±
0.1
0.1
0.2
0.1
0.2
2
3
3
4
II
+267%
± 0.5
0.7
0.2
0.2
0.3
±
0.6 ±
t2.3 ±
t2.4 ±
~
33
12
9
52
112
+833%
±
±
±
±
±
0.5
0.8
0.7
0.5
0.5
0.2
0.2
0.2
0.2
0.1
3
3
2
6
7
+133%
E
4.3
2.0
1.4
3.3
2.9
22
6
4
36
93
+ 1450%
to.4
to.4
0.3
to.3
to.2
Lysosomes
Total
5
6
5
10
18
+20%
[
0.6
to.7
to.6
to.4
0.1
±
±
±
±
±
0.1
0.2
0.2
0.1
0.1
Lipid
t
1.9
0.7
0.06
0.02
0.06
t
0.5
to.3
to.4
[ to.2
0.1
±
±
±
±
±
±
±
±
±
±
1.0
0.1
0.07
0.08
0.03
tl.5
0.2
0.4
[ 3.4
t3.6
[
15
4
0.4
0.3
2
-50%
5
4
4
6
4
0
[
Membrane
Bound
0.1
0.1
0.2
0.2
0.03
4
2
3
4
3
+50%
t
2.4
0.2
0.1
0.1
0.1
±
±
±
±
±
1.0
0.2
0.1
0.1
0.2
0.2
0.1
0.2
0.5
[
l.l
II
I
3
54
139
+ 13,800%
[
t2.8 ±
[ 0.3 ±
[ 0.3 ±
3.1 ±
to. I ±
18
I
I
2
4
+300%
distinguished from cells of the proliferative zone in that
more of their volume was taken up by glycogen, lipid, and
intracellular space. The fact that a larger proportion of the
cells of the proliferative zone was occupied by endoplasmic
reticulum, mitochondria, and Golgi membranes and a lower
proportion was occupied by cytoplasmic space supports the
view that cells of the proliferative zone have higher rates
of metabolism and synthetic function. A high rate of cell
division probably explains the higher cell density in the
proliferative zones and the smaller mean volume of the cells
of the upper proliferative zone relative to reserve-zone cells.
The lower proportion of mitochondria in the cells of the
lower hypertrophic zone suggests that these cells carry on
less aerobic metabolism. The lower proportion of endoplasmic reticulum and Golgi membranes in hypertrophic
cells may indicate that they are less active in synthesis of
matrix molecules than proliferative cells are, and their increased proportion of cytoplasmic space demonstrates that
they accumulate fluid.
By showing that cells of the lower hypertrophic zone
had an increase in their average absolute volume of endoplasmic reticulum, mitochondria, and Golgi membranes, the
results of this study agree with other reports9.2S.26.32 that these
cells remain viable. The high water content of hypertrophiczone cells may explain the reports of BiggersS and Biggers
and Gwatkin6 that freezing destroyed hypertrophic cells,
leaving the other chondrocytes intact. This might occur if
the higher concentration of water in hypertrophic cells alVOL. 68-A, NO.2, FEBRUARY 1986
±
±
±
±
±
Non-Membrane
Bound
21
2
2
63
4
+100%
±
±
±
±
±
4.0
0.4
0.5
3.3
7.0
46
3
8
208
1660
+55,233%
1.3
0.4
0.6
1.9
0.2
6.0
0.5
1.2
t13.2
t42.9
[
8.9
3.0
2.4
t24 .3
t53 .6
±
±
±
±
±
3.3
1.0
1.9
5.2
8.4
67
20
19
493
1992
+ 9860%
Total
7.5
0.5
1.6
16.6
46.5
57
4
II
262
1799
44,875%
11.7
3.3
2.7
27.4
53.7
88
22
21
556
1996
+8972%
Cytoplasmic
Ground Substance
t
t35.3
t40.0
t40.1
41.2
23.3
±
±
±
±
±
4.1
1.6
1.6
5.7
4.6
269
242
259
649
902
+273%
t53.5
[ t46.7
t45.9
38.8
20.4
±
±
±
±
±
2.6
1.5
0.1
4.2
7.6
401
326
365
787
758
+ 133%
Total
100.1%
100.0%
100.0%
100.1%
100.0%
766
605
644
1578
3871
+540%
99.9%
100.7%
99.8%
100.1%
100.2%
750
701
793
2030
3726
+ 432%
lowed the formation of ice crystals that ruptured the cells.
As cell number, volume, and structure changed, matrix
volume and structure changed also. Between the upper proliferative zone and the lower hypertrophic zone, the total
volume of matrix per milliliter declined but the volume of
territorial matrix per milliliter increased and so did the volume of matrix per cell (Table III and Fig. 2). The territorial
matrix volume per cell increased more rapidly between the
upper proliferative zone and the lower hypertrophic zone
than did the volume of the interterritorial matrix per cell
(Table III).
In conventionally fixed tissue, the volume of the territorial matrix per cell increased 14.7 times, from 170 to
2500 cubic micrometers. In tissue fixed with ruthenium
hexamine trichloride it increased 11.2 times, from eightyfive to 950 cubic micrometers. The volume of the interterritorial matrix per cell increased 2.2 times, from 1330 to
2870 cubic micrometers, in conventionally fixed tissue and
1.3 times, from 925 to 1240 cubic micrometers, in ruthenium hexamine trichloride-fixed tissue. These increases
in matrix volume per cell suggest that either the matrix
swelled or the cells synthesized new matrix, or both.
Apparently the cells modified the matrix as they enlarged (Table III). The collagen fibril density of the interterritorial matrix increased slightly between the lower
proliferative zone and the lower hypertrophic zone. This
observation might be explained by Speer's suggestion49 that
the expanding cells compress the matrix and force the trans-
254
1. A. BUCKWALTER ET AL.
physeal collagen fibrils of the longitudinal septa closer together. However, the increase in the collagen-volume
fraction also might be explained by a loss or an alteration
of the non-collagenous matrix components that may accompany preparation of the interterritorial matrix for mineralization lo . II ,5\ accumulation of newly synthesized collagen,
or swelling of the collagen fibrils.
As the volume of the territorial matrix increased, its
collagen fibril density decreased. This observation fits well
with the identification of collagenase activity in the hypertrophic zone, and the suggestion that matrix collagen must
be degraded to allow chondrocyte enlargement I4 ,27 , Since
loss or disruption of the collagen would allow the matrix to
swe1l37 ,38,57, the territorial matrix may have enlarged primarily by accumulating fluid, Cell proliferation, cell enlargement, and matrix synthesis increase physeal volume
and produce longitudinal bone growth. Our results suggest
that swelling of the territorial matrix may also increase physeal volume. The progressive differences in the matrices
among growth-plate zones (Table Ill), combined with the
observations by Dean et al. 14 and Howell et al. 27 and reports
that modification of growth-plate matrix proteoglycans precede mineralization 10, 1 1.54, support the concept that enlarging
chondrocytes synthesize and release enzymes or other factors that modify their matrix,
Chondrocytes enlarged primarily by accumulating water. Our results do not explain how this occurs. It might
result from an injury or a degenerative process that interferes
with the cells' ability to exclude sodium. Soon after an
ischemic insult, kidney, liver, and muscle cells begin to
swe1l 2 ,3,35 ,44 ,55 ,56,58. The nucleus enlarges and decreases in
density, chromatin forms dense clumps, cytoplasmic volume increases, cytoplasmic spaces appear and enlarge, and
the cells deplete their glycogen stores. As these changes
occur, the endoplasmic reticulum and mitochondria dilate;
ribosomes dissociate from the endoplasmic reticulum; distended fragments of endoplasmic reticulum or Golgi membranes appear in the cytoplasm, increasing the volume of
vesicles and vacuoles; and hyaline figures derived from
membranes appear in the cytoplasm and matrix. Although
enlarging chondrocytes showed evidence of glycogen depletion, nuclear and cytoplasmic swelling, and accumulation
of vesicles and vacuoles, they did not show the other
changes found in injured or ischemic cells .
Cell enlargement by fluid accumulation does not necessarily result from injury. For example, plant cells normally
grow by accumulating intracellular fluid l . The enlargement
of growth-plate chondrocytes may be by a similar but specific form of cell growth and development that produces the
sequence of changes in cell structure and function that is
unique to chondrocytes .
Robbins and Cotran43 stressed that cell hypertrophy is
a process whereby "the increased size of cells is due not
to an increased intake of water, called cellular swelling or
edema, but to the synthesis of more ultrastructural components". For more than eighty years, publications describing physeal structure and function have referred to
chondrocyte enlargement as hypertrophy. It is unlikely that
any set of observations will change this practice. However,
for the understanding of growth-plate function it is important
to appreciate that chondrocyte enlargement is not classically
defined hypertrophy, and it may be a specialized form of
cell growth and development that is characterized by the
accumulation of intracellular fluid and associated with modification of the extracellular matrix.
NOTE: The authors (hank Emsl Hunziker for his valuable comments and criticism and Jeancue
Marsh for her assistance in preparing the manuscript.
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