SECTION ONE

EPIPHYSEAL BONE FORMATION

CARL T. BRIGHTON
Organization of the Growth Plate
Blood Supply of the Growth Plate
Structure and Function of the Growth Plate
Cartilage Component
Matrix Component
Fibrous and Fibrocartilaginous Components

The vast majority of the longitudinal growth of a typical mammalian long bone
occurs at the ends of the bone in platelike structures termed growth plates. Both
growth plates, one at the proximal end and the other at the distal end, are
peripheral extensions of the primary center of ossification, a structure that
arises in the midportion of the cartilaginous anlage of the bone-to-be during
early fetal life. Originally the primary center of ossification grows and expands
centrifugally in all directions. However, as it continues to expand,
endochondral bone growth soon becomes confined to the growth plates (Fig. 2-
1).
As growth continues, the growth plates grow away from each other. In each
end of each long bone, at a time characteristic for each species of animal, a
secondary center of ossification, termed the epiphysis, appears. It likewise
grows and expands centrifugally in all directions, although much more slowly
than did the primary center. As the distance between the growth plate and the
epiphysis gradually decreases, the portion of the epiphysis that faces the growth
plate closes and becomes sealed with condensed bone, termed the terminal
bone plate or simply the bone plate.(78) Thereafter the epiphysis assumes a
somewhat flattened hemispheric appearance and slowly fills out the remaining
end of the long bone.
From the above it should be apparent that there is no single acceptable term for
the growth plate proper. "Epiphyseal growth plate" or simply "epiphyseal plate"
is incorrect, for these terms confuse the growth plate with the epiphysis.
Rubin(48) introduced the term "physis" or "physeal segment" in an attempt to
avoid this confusion. Physis, from the Greek word phyo meaning to grow or
growth, is not sufficiently descriptive when referring to such a discrete
morphologic structure as the growth plate. "Physeal segment" is more
descriptive than physis alone, but it is a poor blending of the Greek and the
English. Therefore, in order to avoid confusion and to be grammatically
correct, the term growth plate will be used in this chapter.

ORGANIZATION OF THE GROWTH PLATE
The typical, fully developed growth plate in the mammalian long bone consists
of various tissues acting together as a unit to perform a specialized function
and, as such, is an organ. The specialized function, of course, is longitudinal
growth. Based on tissue content alone, the growth plate may be divided
anatomically into three components: a cartilaginous component, itself divided
into various histologic zones; a bony component, or metaphysis; and a fibrous
component surrounding the periphery of the plate comprising the groove of
Ranvier and the perichondrial ring of LaCroix. How the growth plate
synchronizes chondrogenesis with osteogenesis or interstitial cartilage growth
with appositional bone growth at the same that it is growing in width, bearing
load, and responding to local and systemic forces and factors is a fascinating
phenomenon the key features of which are only beginning to be understood at
the present time. This chapter will discuss the structure and function of the
growth plate in light of those processes that are known.

BLOOD SUPPLY OF THE GROWTH PLATE
Each of the three components of the growth plate has its own distinct blood
supply (Fig. 2-2). (13,16,56) The epiphyseal artery supplies the epiphysis, or
the secondary center of ossification, which itself is not part of the growth plate.
Small branches arise at right angles to the main epiphyseal artery in the
epiphysis and pass through small cartilage canals in the reserve zone to
terminate at the top of the cell columns in the proliferative zone.(56) Each
small branch from the epiphyseal artery arborizes in rakelike fashion to supply
the top portion of from four to ten cell columns. The proliferative zone,
therefore, is well supplied with blood. None of the branches from the
epiphyseal arteries penetrate the cartilage portion of the growth plate beyond
the uppermost part of the proliferative zone; that is, no vessels pass through the
proliferative zone to supply the hypertrophic zone.
The metaphysis is richly supplied with blood both from terminal branches of
the nutrient artery as well as from the metaphyseal arteries arising from the
ascending cervical arteries. The nutrient artery supplies the central region of the
metaphysis, perhaps as much as four fifths of the metaphysis, while the
metaphyseal vessels supply only the peripheral regions of the metaphysis.
Terminal branches from the nutrient and metaphyseal arteries pass vertically
toward the bone-cartilage junction of the growth plate and end in vascular loops
or capillary tufts just below the last intact transverse septa at the base of the
cartilage portion of the plate. The vessels turn back at this level, and venous
branches descend to drain into several veins that eventually terminate in the
large central vein of the diaphysis.(37,41) All (14) or most(3) of the vascular
loops are closed, and microhemorrhages from the vascular loops probably do
not occur. No vessels penetrate the bone cartilage junction beyond the last
intact transverse septa; that is, no vessels pass from the metaphysis into the
hypertrophic zone.
The fibrous peripheral structures of the growth plate, the groove of Ranvier and
the perichondrial ring of LaCroix, are richly supplied with blood from several
perichondrial arteries.

FIG. 2-1 Drawing depicting the two growth plates of a typical long
bone toward the end of fetal life Each growth plate is a peripheral
extension of the original primary center of ossification that arose in
the middle portion of the cartilaginous anlage of the bone-to-be early
in the fetal period.

FIG. 2-2 Drawing showing the blood supply of a typical fully
developed growth plate. (Brighton CT Structure and function of
the growth plate Clin Orthop 136:23 32, l978)
While the metaphysis and the fibrous peripheral components of the growth
plate have an abundant blood supply, only the proliferative zone of the cartilage
portion of the growth plate is adequately supplied with blood. There are no
vessels in the hypertrophic zone of the fully developed growth plate; hence,
that zone is entirely avascular. This avascularity has important implications for
chondrocyte metabolism and matrix calcification.
The blood supply of the human femoral capital growth plate has been studied
thoroughly by Chung16 and, in general, is similar to that described above for a
typical growth plate. Epiphyseal arteries arising from the ascending cervical
branches of the medial and lateral femoral circumflex arteries were the sole
supply of the epiphysis in 82% of the femoral heads studied by Chung.
However, in 28% of the femoral heads, the epiphysis was also supplied by one
or two branches of the artery of the ligamentum teres femoris (Fig. 2-3). The
metaphysis is supplied with blood from terminal branches of the nutrient artery
as well as from metaphyseal arteries arising from the ascending cervical
arteries. The fibrocartilaginous peripheral structure of the femoral capital
growth plate is richly supplied with blood from several perichondrial arteries
arising from the ascending cervical arteries.

STRUCTURE AND FUNCTION OF THE GROWTH PLATE CARTILAGE
COMPONENT
The cartilage portion of the growth plate begins at the top of the reserve zone
and ends with the last intact transverse septa at the bottom of the cell columns
in the hypertrophic zone. It has been divided into various zones according to
morphology or function (Fig. 2-4). The reserve zone begins just beneath the
secondary bony epiphysis, followed by the proliferating zone and the
hypertrophic zone. The hypertrophic zone is sometimes further subdivided into
the zone of maturation, the zone of degeneration, and the zone of provisional
calcification.

FIG. 2-3 Drawing showing the blood supply of the capital
femoral growth plate. (RZ = reserve zone; PZ = proliferative
zone; HZ = hypertrophic zone)

RESERVE ZONE
The reserve zone lies immediately adjacent to the secondary bony epiphysis.
Various terms have been applied to this zone, including resting zone, zone of
small-size cartilage cells, and germinal zone. However, these cells are not
resting, are not small in comparison with the cells in the proliferative zone, and
they are not germinal cells. They appear to store lipid and other materials and
perhaps are held in reserve for later nutritional requirements. If that is true, the
term reserve zone may be appropriate. The cells in this zone are spherical, exist
singly or in pairs, are relatively few when compared with the number of cells in
other zones, and are separated from each other by more extracellular matrix
than are cells in any other zone. The cells in the reserve zone are approximately
the same size as the cells in the proliferative zone.(12) The cytoplasm exhibits a
positive staining reaction for glycogen. Electron microscopy reveals these cells
to contain abundant endoplasmic reticulum, a clear indication that they are
actively synthesizing protein. They contain more lipid bodies and vacuoles than
do cells in other zones(12) but contain less glucose-6-phosphate
dehydrogenase, lactic dehydrogenase, malic dehydrogenase, and
phosphoglucoisomerase. (29) The zone also contains the lowest amount of
alkaline and acid phosphatase,(30) total and inorganic phosphate, calcium,
chloride, potassium, and magnesium.(58) The matrix in the reserve zone
contains less lipid,(24)glycosaminoglycan, protein polysaccharide, moisture,
and ash(35) than the matrix in any other zone. It exhibits less incorporation of
radiosulfur (35S) than any other zone and also shows less Iysozyme activity
than the other zones.(51) It contains the highest content of hydroxyproline of
any zone in the plate. (24) Collagen fibrils in the matrix exhibit random
distribution and orientation. Matrix vesicles are also seen in the matrix, but they
are fewer than in other zones. The matrix shows a positive histochemical
reaction for the presence of a neutral mucopolysaccharide or an aggregated
proteoglycan.
Measurements of oxygen tension in the extracellular space in the different
zones of the growth plate reveal PO2 to be low (20 5 + 2.1 mm Hg) in the
reserve zone.(7) This probably indicates that the blood vessels that pass through
this zone in cartilage canals to arborize at the top of the proliferative zone do
not actually supply the reserve zone itself.
The chondrocytes in the reserve zone either do not proliferate or do so only
sporadically. Therefore, the zone is not a germinal layer containing the so-
called mother cartilage cells. As a matter of fact, the function of the zone is not
clear. The high lipid body and vacuole content may mean that these materials
are being stored for later nutritional requirements. and in this sense, the
function of the zone is one of storage.

FIG. 2-4 Drawing depicting the various zones of the
cartilaginous portion of the growth plate. (Brighton CT
Structure and function of the growth plate. Clin Orthop
136:23 32, 1978)

PROLIFERATIVE ZONE
The spherical, single or paired chondrocytes in the reserve zone give way to
flattened chondrocytes in the proliferative zone. They are aligned in
longitudinal columns with the long axis of the cells perpendicular to the long
axis of the bone. The cytoplasm stains positively for glycogen. Electron
microscopy shows the chondrocytes to be packed with endoplasmic
reticulum.(12,20) Point-counting analysis reveals the percent of the
cytoplasmic area occupied by endoplasmic reticulum to increase from 14.9% at
the top of the zone to 40.1% at the bottom of the zone. (12) Biochemical
analyses reveal the zone of proliferation to contain the highest content of
hexosamine, (19, 24) inorganic pyrophosphate,(30) and sodium, chloride, and
potassium.(53) The proliferating zone shows the highest incorporation of 35S
of any zone in the growth plate, and it also has the highest level of Iysozyme
activity.(5l)
Tritiated thymidine autoradiographic studies have indicated that the
chondrocytes in the proliferative zone are, with few exceptions, the only cells
in the cartilage portion of the growth plate that divide.(25) The top cell of each
column is the true "mother" cartilage cell for each column, and it is the
beginning or the top of the proliferating zone that is the true germinal layer of
the growth plate. Longitudinal growth in the growth plate is equal to the rate of
production of new chondrocytes at the top of the proliferating zone multiplied
by the maximum size of the chondrocytes at the bottom of the hypertrophic
zone.(53) Kember (25) showed that the average number of new chondrocytes
produced daily in each column in the growth plate of the proximal tibia in the
rat was five. Since the average diameter of the chondrocyte at the bottom of the
hypertrophic zone is about 30 um, the rate of growth from that particular
growth plate is about 150 um/day. He further calculated that each division of a
top cell in a cell column contributes 29 cells.(26) This means that each division
of a top cell eventually contributes 0.9 mm of longitudinal growth to the rat
tibia (29 x 30 um = 870 um). Complete growth of the rat tibia would require 40
to 50 top-cell divisions. These principles (but not the absolute numbers)
presumably hold true for all mammalian growth plates.
The matrix of the proliferating zone contains collagen fibrils, distributed at
random, and matrix vesicles, confined mostly to the longitudinal septa. The
matrix shows a positive histochemical reaction for a neutral
mucopolysaccharide or an aggregated proteoglycan.
Oxygen tension is higher in the proliferating zone (57 +/- 5.8 mm Hg) than in
any of the other zones of the growth plate.(7) This is due to the rich vascular
supply present at the top of the zone. Considering the relatively high oxygen
tension coupled with the presence of glycogen in the chondrocytes, it is
apparent that aerobic metabolism with glycogen storage is occurring.
Thus the function of the proliferative zone is twofold: matrix production and
cellular proliferation. The combination of these two functions equals linear or
longitudinal growth. It is a paradox that while this chondrogenesis or cartilage
growth is solely responsible for the increase in linear growth of the long bone,
the cartilage portion of the plate itself does not increase in length. This, of
course, is due to the vascular invasion that occurs from the metaphysis with the
resultant removal of chondrocytes at the bottom of the hypertrophic zone,
events that, in the normal growth plate, exquisitely balance the rate of cartilage
production.

HYPERTROPHIC ZONE
The flattened chondrocytes in the proliferative zone become spherical and
greatly enlarged in the hypertrophic zone. These changes in cell morphology
are quite abrupt, and one can usually determine the end of the proliferative zone
and the beginning of the hypertrophic zone within an accuracy of one to two
cells. By the time the average chondrocyte reaches the bottom of the
hypertrophic zone, it has enlarged some five times over what its size was in the
proliferative zone.(12) The cytoplasm of the chondrocytes in the top half of the
hypertrophic zone stains positively for glycogen (periodic acid-Schiff [PAS]
reaction coupled with diastase digestion), but near the middle of the zone the
cytoplasm abruptly loses this ability. On light microscopy, the chondrocytes in
the hypertrophic zone appear vacuolated. Toward the bottom of the zone, such
vacuolation becomes extensive, nuclear fragmentation occurs, and the cells
appear nonviable. At the very bottom of each cell column the lacunae appear
empty and are devoid of any cellular content.
On electron microscopy the chondrocytes in the top half of the hypertrophic
zone appear normal and contain the full complement of cytoplasmic
components. (12,21) However, in the bottom half of the zone, the cytoplasm
contains holes that occupy over 58% of the total cytoplasmic
column. (12) Obviously, it is holes and not vacuoles that account for the
"vacuolation" seen on light microscopy. Electron microscopy also shows that
glycogen is abundant in the chondrocytes in the top half of the zone, diminishes
rapidly in the middle of the zone, and disappears completely from the cells in
the bottom portion of the zone. The last cell at the base of each cell column is
clearly nonviable and shows extensive fragmentation of the cell membrane and
the nuclear envelope with loss of all cytoplasmic components except a few
mitochondria and scattered remnants of endoplasmic reticulum. Clearly, the
ultimate fate of the hypertrophic chondrocyte is death.
Electron micrographs reveal electron-dense granules in mitochondria of growth
plate chondrocytes.(35,39) These granules are not removed by
microincineration(39) (and hence, are mineral) and have been shown by direct
analysis to have the characteristic radiographic spectra of calcium and
phosphorus.(55) They are present in highest concentration in chondrocytes in
the hypertrophic zone in the normal growth plate and are absent or greatly
diminished in number in the rachitic growth plate.(49) Histochemical
localization of calcium at the ultrastructural level shows the mitochondria and
cell membranes of chondrocytes in the top half of the hypertrophic zone to be
loaded with calcium (Fig. 2-5). (8,9) Toward the middle of the zone,
mitochondria rapidly lose calcium, and at the bottom of the zone, both
mitochondria and cell membranes are totally devoid of calcium. All of these
studies cited above provide circumstantial evidence that mitochondrial calcium
may be involved in cartilage calcification.

FIG. 2-5 Electron micrographs of mitochondria from the hypertrophic
zone of the growth plate stained for calcium with potassium
pyroantimonate. (A) Top portion of the hypertrophic zone stained
conventionally. (B) Top portion of the hypertrophic zone showing
positive stain in mitochondria. (C) Middle portion of the hypertrophic
zone showing beginning of depletion of calcium stain from the
mitochondria. (D) Bottom of hypertrophic zone showing almost
complete depletion of mitochondrial calcium. (original magnification
x 82,000)
Biochemical analyses of the hypertrophic zone indicate that this region, or at
least the upper three fourths of it, is active metabolically. It contains the highest
content of alkaline phosphatase, acid phosphatase, glucose-6-phosphate
dehydrogenase, lactic dehydrogenase, malic dehydrogenase and
phosphoglucoisomerase;(29,30) total and inorganic phosphate, calcium, and
magnesium;(58) moisture and ash;(35) and lipid.(24) It contains the lowest
content of hydroxyproline(24) and hexosamine(19)
Oxygen tension in the hypertrophic zone is quite low (24.3 +/- 2.4 mm
Hg).(7) No doubt this low oxygen tension is due to the avascularity of the zone.

FIG. 2-6 Drawing summarizing the metabolic events occurring
in the various zones of the growth plate. (Brighton CT, Hunt
RM: The role of mitochondria in growth plate calcification as
demonstrated in a rachitic model. J Bone Joint Surg 60A:630
639, 1978)
A summary of the metabolic events occurring in the cartilage portion of the
growth plate, or, more correctly, in the proliferative and hypertrophic zones, is
presented in Figure 2-6. (10) In the proliferative zone, oxygen tension is high,
aerobic metabolism occurs, glycogen is stored, and mitochondria form
adenosine triphosphate (ATP). Mitochondria can form ATP or store calcium,
but they cannot do both at the same time.(33) Thus, ATP formation and
calcium accumulation are alternative processes rather than simultaneous ones.
In the proliferative zone, the energy requirement for matrix production and
cellular proliferation is high, and mitochondria form ATP. In the hypertrophic
zone, oxygen tension is low, anaerobic metabolism occurs, and glycogen is
consumed until, near the middle of the zone, it is completely depleted. In the
top half of the hypertrophic zone, mitochondria switch from forming ATP to
accumulating calcium.(3) Why this switch occurs at this level in the growth
plate is not entirely clear. However, both the formation of ATP and calcium
accumulation are active processes requiring energy.(33)Such energy comes
from the respiratory chain in the mitochondria. ATP formation requires, in
addition, the presence of adenosine diphosphate (ADP), while calcium
accumulation does not. It may well be that in the hypertrophic zone there
simply is not enough ADP to provide for significant ATP formation. In any
event, mitochondria in the top half of the hypertrophic zone accumulate
calcium and do not form ATP.
In the bottom half of the hypertrophic zone, as stated above, glycogen is
completely depleted. In this area of low oxygen tension, there is no other
source of nutrition to serve as an energy source for the mitochondria. Since
retention of calcium by mitochondria (as well as uptake of calcium) is an active
process requiring energy,(34) as soon as the glycogen supply of the
chondrocytes is exhausted, mitochondria release calcium. This released calcium
may play a role in matrix calcification (see below).
The matrix of the hypertrophic zone, unlike the other zones, shows a positive
histochemical reaction for an acid mucopolysaccharide or a disaggregated
proteoglycan. Electron microscopy reveals that there is a progressive decrease
in length of proteoglycan aggregates and a decrease in the number of subunits
of the aggregates in the matrix as one progresses from the reserve zone through
the hypertrophic zone.(15) The distance between the subunits increases at the
same time. It is speculated by some that the large proteoglycan aggregates with
tightly packed subunits may inhibit mineralization or the spread of
mineralization, whereas smaller aggregates with widely spaced subunits at the
bottom of the hypertrophic zone may be less effective in preventing mineral
growth.(15) Lysozyme may be involved in the disaggregation of large
proteoglycan aggregates,(28,42,51) or Iysosomal enzymes, especially neutral
proteases, may degrade the proteoglycan.(49) In any event, it seems apparent
that proteoglycan disaggregation or degradation must occur before there can be
significant mineralization.(27,23)
The initial calcification ("seeding" or "nucleation") that occurs in the growth
plate in the bottom of the hypertrophic zone (zone of provisional calcification)
does so within or upon matrix vesicles that are present in the longitudinal septa
of the matrix (Fig. 2-7).(1,4,5,18)Matrix vesicles are very small structures
(lOOOA-1500A in diameter) that are enclosed in a trilamellar membrane and,
therefore, are produced by the chondrocyte. They occur in greatest
concentration in the hypertrophic zone.(4) Matrix vesicles are rich in alkaline
phosphatase,(2) and this enzyme may act as a pyrophosphatase to destroy
pyrophosphate, another inhibitor of calcium phosphate
precipitation.(18) Matrix vesicles begin to accumulate calcium at the same
level in the middle of the hypertrophic zone at which mitochondria begin to
lose calcium (Fig. 2-8).(5-9) This is circumstantial evidence indicating that
mitochondrial calcium is involved in the initial calcification that occurs in the
growth plate. The initial calcification in the matrix vesicles may be in the form
of amorphous calcium phosphate,(43) but this rapidly gives way to
hydroxyapatite crystal formation. With crystal growth and confluence, the
longitudinal septa become calcified. This occurs in the bottom portion of the
hypertrophic zone, a region frequently called the zone of provisional
calcification.
This calcification makes the intercellular matrix relatively impermeable to
metabolites. Diffusion coefficients of the various zones of the growth plate
have been measured, and the hypertrophic zone has the lowest diffusion
coefficient in the entire growth plate.(54) This is due primarily to the high
mineral content of that zone, and suggests the following sequence of events:
1. Calcification occurs.
2. Diffusion of nutrients and oxygen to the hypertrophic chondrocyte is
decreased.
3. Anaerobic glycolysis with glycogen consumption occurs until all the
glycogen is depleted.
4. Mitochondria release calcium.
5. Nucleation occurs in the matrix vesicles.
6. Calcification of the matrix occurs.

FIG. 2-7 Electron micrographs of matrix vesicles from the various
zones of the growth plate stained for calcium with potassium
pyroantimonate. (A) Proliferative zone stained conventionally. (B)
Proliferative zones stained with pyroantimonate (negative stain). (C)
Middle of hypertrophic zone shows clumps of calcium-antimonate
complex upon or within matrix vesicles. (D) Bottom of hypertrophic
zone shows crystal formation obliterating the matrix vesicle. (original
magnification x 180,000).
Thus, a cycle is established that results ultimately in the death of the
hypertrophic chondrocyte (Fig. 2-9).
The functions of the hypertrophic zone seem clear: to prepare the matrix for
calcification and to calcify the matrix. Although these processes are complex
biophysical phenomena, it is evident from the studies cited above that we are
beginning to unravel the mechanisms and factors controlling matrix
calcification.

BONY COMPONENT (METAPHYSIS)
The metaphysis begins just distal to the last intact transverse septum at the base
of each cell column of the cartilage portion of the growth plate (Fig. 2-10). It
ends at the point at which narrowing or funnelization of the bone end ceases,
that is, where the wider metaphysis meets the narrower diaphysis.(48) In the
first part of the metaphysis just distal to the cartilage portion of the plate, the
oxygen tension is low (19.8 + 3.2 mm Hg).(7) The low oxygen tension, as well
as the rouleaux formation of the red cells frequently seen just distal to the last
intact transverse septa,(6) indicates that this is a region of vascular stasis. A
flocculent, electron-dense material present within the lumen of vascular sprouts
invading the transverse septa may likewise indicate the presence of circulatory
stasis within these vessels.(50) Also, high levels of phosphoglucoisomerase, an
enzyme active in anaerobic metabolism, are found in this region and are
compatible with vascular stasis(29)
In the first part of the metaphysis, the first lacuna distal to the last intact
transverse septum at the base of each column of cells is, by light microscopy,
either empty or contains one or more red cells. Electron microscopy shows
capillary sprouts or loops lined by a layer of endothelial and perivascular cells
invading the base of the cartilage portion of the plate.(50) Cytoplasmic
processes from these cells push into the transverse septa and, presumably
through Iysosomal enzyme activity, degrade and remove the nonmineralized
transverse septa. At this same level in the metaphysis, the longitudinal septa are
partially or completely calcified. Osteoblasts, which are plump, oval-shaped
cells with eccentric nuclei, line up along the calcified bars. Between the
osteoblasts lining the calcified bars and the capillary sprouts are seen
osteoprogenitor cells, cells with little cytoplasm but with a prominent ovoid to
spindle-shaped nucleus(27) This region of vascularized calcified cartilage with
little or no bone formation occurring on the calcified bars is termed the primary
spongiosum.(36)

FIG. 2-8 Montage of electron micrographs of the hypertrophic zone
shows that the calcium-antimonate staining is predominantly
intracellular at the top of the zone but becomes progressively more
extracellular toward the bottom of the zone. The inserts on the right are
of mitochondria in chondrocytes at corresponding levels in the zone.
Note the gradual loss of the calcium stain the farther down the zone the
mitochondrion is located. Inserts on the left are of matrix vesicles at
corresponding levels in the zone. Note the gradual accumulation of the
calcium stain the farther down the zone the vesicle is located. (Brighton
CT, Hunt RM: Mitochondral calcium and its role in calcification. Clin
Orthop 100:406 416, 1974)

FIG. 2-9 Drawing showing events in the hypertrophic zone
relating matrix calcification to decrease pO2, glycogen
metabolism, and mitochondrial, calcium release (Brighton CT,
Hunt RM: The role of mitochondria in growth plate
calcification as demonstrated in a rachitic model. J Bone Joint
Surg 60A:630 638, 1978)

FIG. 2-10 Drawing of the metaphysis showing the interlocking of the
primary and secondary spongiosa. (Brighton CT: Structure and
function of the growth plate. Clin Orthop 136:23-32, 1978)
A short distance (within a cell or two) further down the calcified longitudinal
septa, the osteoblasts begin laying down bone (termed endochondral
ossification, i.e., bone formation within or upon cartilage). The further down or
into the metaphysis one progresses, the more bone is formed on the calcified
cartilage bars. At the same time, the bars gradually diminish in thickness until
they disappear altogether. This region, where bone is laid down on calcified
cartilage bars, is termed the secondary spongiosum.(36)
Still further down in the metaphysis, the fiber bone that was formed originally
is replaced with lamellar bone. This gradual replacement of the calcified
longitudinal septa with newly formed fiber bone, as well as the gradual
replacement of fiber bone with lamellar bone, is termed internal or histologic
remodeling. (32)
Large, irregularly shaped cells with foamy, eosinophilic cytoplasm and one or
more nuclei each containing several nucleoli are evenly distributed throughout
the entire metaphysis except in the primary spongiosum. These osteoblasts are
also seen subperiosteally around the outside of the metaphysis where it
diminishes in diameter to meet the diaphysis. This narrowing or funnelization
of the metaphysis is termed external or anatomic remodeling.(32)
The functions of the metaphysis therefore, are vascular invasion of the
transverse septa at the bottom of the cartilaginous portion of the growth plate,
bone formation, and remodeling, both internal and histologic (removal of
calcified cartilage bars and replacement of fiber bone with lamellar bone) and
external or anatomic (funnelization of the metaphysis).

FIBROUS AND FIBROCARTILAGINOUS COMPONENTS
Encircling the typical long-bone growth plate at its periphery are a wedge-
shaped groove of cells, termed the ossification groove, and a ring or band of
fibrous tissue and bone, termed the perichondrial ring (Fig. 2-11).
Ranvier,(46) the first to describe these structures, concentrated his study on the
cells in the groove, which is now named after him. LaCroix(31) studied the
perichondrial ring in detail, and this structure is frequently named after him.
Although it is true that the ossification groove and the perichondrial ring are
simply different parts of the same structure, they do have different functions,
and for that reason alone it is advantageous to consider them as separate and
distinct entities. These structures will be described first as they are found in the
typical long-bone growth plate (distal femur) and then as they exist in a
somewhat different form in the femoral capital growth plate.

FIG. 2-11 Photomicrograph of the periphery of the distal femoral
growth plate of a 14-day-old rat shows the ossification groove of
Ranvier (A) and the perichondrial ring of LaCroix (B). (H&E x loo)
(Brighton CT: Clinical problems in epiphyseal plate growth and
development, pp 107-113. AAOS Instructional Course Lecture, vol
XXIII, p 107. St Louis, CV Mosby, 1974)
The ossification groove contains round to oval cells that, on light microscopy,
seem to flow from the groove into the cartilage at the level of the beginning of
the reserve zone. For that reason, and since these cells avidly incorporate
tritiated thymidine, it appears that the function of the groove of Ranvier is to
contribute chondrocytes to the growth plate for the growth in diameter, or
latitudinal growth, of the plate.(52) In a recent, definitive study using electron
microscopy and autoradiography, three groups of cells were identified in the
ossification groove: a group of densely packed cells that seemed to be
progenitor cells for osteoblasts that form the bony band in the perichondrial
ring; a group of undifferentiated cells and fibroblasts that contribute to
appositional chondrogenesis and, hence, growth in width of the growth plate;
and fibroblasts amid sheets of collagen that cover the groove and firmly anchor
it to the perichondrium of the hyaline cartilage above the growth plate.(57)

FIG. 2-12 Photomicrograph of the periphery of the proximal femoral
growth plate of a 14-day-old rat shows the fibrocartilaginous structure
that replaces the groove of Ranvier and perichondrial ring in other
growth plates. (H&E x 100)
The perichondrial ring is a dense fibrous band that encircles the growth plate at
the bone-cartilage junction and in which collagen fibers run vertically,
obliquely, and circumferentially.(45) It is continuous at one end with the group
of fibroblasts and collagen fibers in the ossification groove and at the other end
with the periosteum and subperiosteal bone of the metaphysis. In rodents,
rabbits, and dogs, the innermost layer of the perichondrial ring consists of bone
that may or may not be attached to the subperiosteal bone of the metaphysis.
This cylindrical sheath of bone may not be present in all species at all ages in
all growth plates. For instance, it is not present in the proximal femur in the
human at any age. " Whether or not bone is present in the perichondrial ring,
there is no doubt that the ring provides mechanical support for the otherwise
weak bone-cartilage junction of the growth plate (17,47,52)
Hence the function of the ossification groove is to provide chondrocytes for the
growth in width of the growth plate, and the function of the perichondrial ring
is to act as a limiting membrane that provides mechanical support to the growth
plate.
In the femoral capital growth plate, the functions of the ossification groove and
the perichondrial ring are the same as in any typical long-bone growth plate,
but the structure of these peripheral tissues is quite different. Instead of a rather
distinct ossification groove and perichondrial ring, these two structures are
replaced by one structure that consists of fibrocartilage in the area that is
occupied by the groove of Ranvier and the perichondrial ring in other growth
plates (Fig. 2-12). This structure apparently has the same functions as the
groove of Ranvier and the perichondrial ring, that is, to provide for latitudinal
growth of the growth plate (top portion of the fibrocartilaginous structure) and
to provide mechanical support to the growth plate (remainder of the
fibrocartilaginous structure).(17)


SECTION TWO
DIAPHYSEAL BONE FORMATION
ARTHUR W. FETTER

One of the earliest indications that the cartilage model of a long tubular bone is
about to be replaced is the appearance of a thin collar (ring or sleeve) of
osseous tissue around the middle. The formation of the osseous collar is
induced when the cartilage cells in the area mature and hypertrophy, and the
matrix they have produced becomes mineralizable. The collar of bone, which
represents the first stage in the development of the cortex of the evolving bone,
is formed through intramembranous ossification from osteoblasts in the inner or
osteogenic layer of the perichondrium. This fibrous tissue layer, because of its
location, should be called periosteum. The cortical shell consists of a loosely
woven matrix with more cells per unit area than that which occurs in mature
bone.
The formation of the periosteal osseous collar is rapidly followed by the
extension of vascular buds from the periosteum through the cortical shell into
the cartilaginous area. This process is induced by the degeneration and death of
cartilage cells. These primitive vessels enter the degenerating cartilage,
bringing with them mononuclear cells and mesenchymal elements, which give
rise to the chondroclasts, osteoclasts, and osteoblasts. These cells are essential
in the process of enchondral ossification and the formation of the hematopoietic
marrow of the evolving bone.
As the diaphysis elongates, the cortical shell of bone continues to develop
adjacent to the growth plate and remains at, or slightly beyond, the level of the
zone of hypertrophied cartilage cells, providing structural support for the
cartilaginous epiphysis. This extension of the cortical shell forms a three-
dimensional cup and is called Ranvier's ossification groove (encoche
deRanvier). In order to maintain the funnel shape of the bone as it grows in
length, remodeling of the metaphysis begins almost as soon as any bone is
formed.
During the rapid advance of enchondral ossification toward the epiphyseal ends
of the elongating young bone, growth is also occurring subperiosteally in the
diaphysis. The periosteum becomes quite thick, and spicules of new bone are
deposited more or less perpendicularly between the cambium layer of the
periosteum and the original cortical shell. The matrix of the rapidly deposited
bone is loosely woven, resembling burlap, and contains many cells per unit area
of bone. As cortical growth in the area of the diaphysis slows, osteoblasts line
up on the surface of the bone spicules and slowly deposit lamellar bone in the
form of an "inlay" into the spaces between the trabeculae. This filling between
the trabeculae results in the formation of a solid cortex. At this stage, the cortex
consists of a mixture of woven fiber bone with its haphazard fibrillar
arrangement and numerous osteocytes, and the lamellar bone with a parallel
arrangement of collagen fibers and fewer osteocytes. The inlay bone has an
osteonal appearance, but true osteons are formed as refill after osteoclasts have
cut a hole in cortical bone. Therefore, the inlay bone does not qualify as true
osteonal bone.









SECTION THREE
CORTICAL REMODELING
ARTHUR W. FETTER

Once a cortical surface is formed, layer after layer of bone is deposited on the
surface in the form of coarse lamellar bone. Since this bone is not initially
deposited along lines of stress, but rather in response to vascular patterns and
growth patterns, remodeling must occur. This remodeling is accomplished by
osteoclasts, the number and activity of which are influenced by circulatory,
metabolic, and mechanical factors.(59) The initial event in remodeling is the
removal by osteoclasts of matrix that was previously deposited by osteoblasts.
This occurs through the action of a "cutting cone" of osteoclasts, which
progresses more or less longitudinally along the developing shaft of the bone.
The cutting cone advances along the course of the vessel, producing a
resorption cavity (Fig. 2-13, A). Following along behind the cutting cone,
osteoblasts become aligned on the walls of the resorption cavity and secrete a
layer of mucopolysaccharides on the surface of the cavity, which is referred to
as a cement or reversal line. Successive generations of osteoblasts then produce
layers of bone that fill in the resorption cavity in centripetal fashion, leaving a
small central canal that contains vessels and nerves. This process results in the
production of an osteon or haversian system, which consists of the central
canals with their blood vessels, canaliculi, and the concentric lamellae of
bone (Fig. 2-13, B). The canaliculi contain osteocytic processes during life and
radiate from the central canal, thereby interconnecting the osteocytic lacunae.
Thus, each haversian system or osteon is a single metabolic unit nourished by
the vessel in the central canal, with nourishment proceeding outward through
the canaliculi (Fig. 2-14).

FIG. 2-13 (A) Numerous resorption cavities in various
stages of refill in cortical bone. Note recently formed
resorption cavities (short arrows) and others that are
partially filled by osteonal bone formation (long arrows)
The dark circumferential bands within the resorption
cavities are osteoid seams of the developing osteon. The
mineralized section of bone is stained with Goldner's
modified trichrome stain. (B) Mature cortical bone
characterized by fully developed osteons with minimal
resorptive activity. The mineralized section of bone is
stained with Goldner's modified trichrome stain.

FIG. 2-14 A single osteon characterized by a central canal
(C) osteocytic lacunae (O), and osteocytic processes within
canaliculi (arrows). The mineralized bone section is
stained with basic fuchsin.

FIG. 2-15 Cortical bone consisting of numerous osteons
(O) and interstitial fragments (F). Unstained section of
mineralized bone.
As cutting cones remodel bone, they do so with a "skew" determined by
mechanical stresses. Therefore, the cutting cone may cross osteonal lines,
removing portions of several osteons. When these resorption cavities are
refilled, portions of the original osteons are left behind. After several waves of
such activity, numerous interstitial fragments remain. Since a cement or
reversal line is deposited following the formation of the cavity, the interstitial
fragments are not directly connected to any haversian blood supply (Fig. 2-15).
With the passage of years and many waves of remodeling activity, the number
of interstitial fragments becomes greater and greater. Volkmann's canals course
transversely through the cortex, connecting adjacent haversian systems and
providing intercommunication between the medullary and the periosteal blood
supply. These canals are not surrounded by concentric layers of bone, as are the
osteons.



















Endochondral Bone Formation
Author: David Abbasi
Topic updated on 09/08/13 7:50pm



Introduction
Enchondral bone formation occurs in
o longitudinal physeal growth
o embryonic long bone formation
o non-rigid fracture healing (secondary healing)
Cell biology
o enchondral bone formation occurs with a cartilage model
chondrocytes produce cartilage which is absorbed by osteoclasts
osteoblasts lay down bone on cartilaginous framework (bone replaces cartilage,
cartilage is not converted to bone)
forms primary trabecular bone
bone deposition occurs on metaphyseal side
type X collagen associated with enchondral ossification
Molecular biology
o chondrocytes play a critical role in endochondral bone formation throughout the
formation of the cartilage intermediate
o transcription factors involved in regulation of chondrocytes include
Sox-9
considered a major regulator of chondrogenesis, regulates several
cartilage-specific genes during endochondral ossification, including
collagen types II, IV, and XI and aggrecan
PTHrP
delays differentiation of chondrocytes in the zone of hypertrophy
Biomechanics
o variables that affect growth across the physis
Hueter-Volkmann Law
compression across the growth plate slows longitudinal growth
tension accerelates longitudinal growth
Anatomy
Blood supply
o perichondrial artery
You have not been heard from for a while.
major source of nutrition to physis
Longitudinal Physeal Growth

Physeal Growth Plate
(letters on left correspond to histology in top right)
Reserve
Zone
Cells store lipids, glycogen, and
proteoglycan aggregates for later growth
and matrix production
Low oxygen tension

Gaucher's
diastrophic dysplasia
Kneist*
Pseudoachondroplasia*
Proliferative
Zone
Proliferation of chondrocytes with
longitudinal growth and stacking of
chondrocytes.
Highest rate of extracellular matrix
production
Increased oxygen tension in
surroundings inhibits calcification

Achondroplasia
Gigantism
MHE
Hypertrophic
Zone
Zone of chondrocyte
maturation, chondrocyte hypertrophy,
and chondrocyte calcification.
Three phases occur in the hypertrophic
zone
o Maturation zone: preparation of
matrix for calcification,
chondrocyte growth
o Degenerative zone: further
preparation of matrix for
calcification, further chondrocyte
growth in size (5x)
o Provisional calcification
zone: chondrocyte death allows
calcium release, allowing
calcification of matrix
Chondrocyte maturation regulated by
local growth factors (parathyroid related
peptides, expession regulated by Indian
hedgehog gene)
Type X collagen produced by
hypertrophic chondrocytes important for
mineralization
SCFE (not renal)
Rickets (provisional calcification
zone)
Enchondromas
Mucopolysarcharide disease
acromegaly
SED
MED
Schmids
Kneist*
Pseudoachondroplasia*
Fractures most commonly occur
through the zone of provisional
calcification, specifically Salter-
Harris I fractures
Primary Spongiosa
(metaphysis)
Vascular invasion and resportion of
transverse septa.
Osteoblasts align on cartilage bars
produced by physeal expansion.
Primary spongiosa mineralized to form
woven bone and then remodels to
become secondary spongiosa (below)
Metaphyseal "corner fracture" in
child abuse
Scurvy
Secondary
spongiosa
(metaphysis)
Internal remodeling (removal of cartilage
bars, replacement of fiber bone with
lamellar bone)
External remodeling (funnelization)
Renal SCFE
Physis Periphery
Groove of
Ranvier
During the first year of life, the zone
spreads over the adjacent metaphysis to
form a fibrous circumferential ring
bridging from the epiphysis to the
diaphysis.
This ring increases the mechanical
strength of the physis and is responsible
for appositional bone growths
o supplies chondrocytes to
periphery

Osteochondroma
Perichondrial
fibrous ring of
La Croix
Dense fibrous tissue that is the primary
limiting membrane that anchors
and supports the physis through
peripheral stability



Embryonic Long Bone Formation
Overview
o allows growth in width and length
o formed from mesenchymal anlage around 6th week in utero.
Steps of formation include
o vascularization
vascular buds invade the mesenchymal model
o primary ossification centers form
(at ~ 8 weeks) osteoprogenitor cells migrate through vascular buds and
differentiate into osteoblasts forming the primary ossification centers
o cartilage model forms
grows through appositional (width) and interstitial (length) growth
o marrow forms
marrow is formed by resorption of central portion of the cartilage anlage
by myeloid precursor cells that migrate in through the vascular buds
o secondary ossification centers form
develop at bone ends and lead to epiphyseal ossification center (growth
plate)
Non-Rigid Fracture Healing
Overview
o mechanism of bone formation is similar to physeal enchondral ossification
Cell biology
o soft callus is the cartilage intermediate
o bone replaces callus via same chondrocyte proliferation, chondrocyte hypertrophy,
and finally chondrocyte calcification
Examples include
o casting and bracing
o intramedullary nailing
allows for motion at the fracture site, which promotes bone formation both
directly (intramembranous ossification) and through a cartilage intermediate
(endochondral ossification)