Copynghi

Pathogenic
BY

984 by The Journal

of

thaw and Joint Surgery,

Mechanisms
VICTOR

STANESCU,
AND

From

PIERRE

in Osteochondrodysplasias

M.D.

D.M.SCI.*,

MAROTEAUX,

the Unite de Recherches

H#{243}pitaldes

with

nineteen

different

forms

of human

osteo-

chondrodysplasia.
Cartilage
biopsies were obtained
during orthopaedic
procedures.
Postmortem
specimens
were obtained
within a short time after death. The combined morphological
and biochemical
studies
revealed
specific
abnormalities
suggestive
of a particular
biochemical
defect in several chondrodysplasias.
In pseudoachondroplasia,
non-collagenous
protein
accumulated
in the rough
endoplasmic
reticulum
of
chondrocytes

and

a proteoglycan

species

that

M.D.*,

normally

eases

that

in this

syndrome

FRANCE

Paris

in several
atypical
cases of spondyloepiphyIn a pair of twins with an atypical
form
of spondyloepiphyseal
dysplasia,
the presence
of many
multinucleated
chondrocytes
suggested
a primary
impairment
of cell division.
CLINICAL
RELEVANCE:
A knowledge
of the pathogenic mechanisms
in osteochondrodysplasias
might improve the classification;
aid in diagnosis,
prognosis,
and
genetic counseling;
and contribute
to the understanding
of normal
endochondral
growth.
The data that we obtamed indicate
the basic biochemical
defect that may be
present
in some of the chondrodysplasias,
and provide
a rational
basis for setting conceptual
directions
for reseal

teoglycan.

suggested

PARIS,

dysplasia.

in this
Most

area.

osteochondrodysplasias

that

are due

of bone growth
and
cartilaginous
growth

lysosomal

various

of

proteoglycans

in

chondrocytes

were found
in spondylometaphyseal
dysplasia
of the
Kozlowski
type.
An abnormal
organization
of type-Il
collagen
was
found in fibrochondrogenesis.
In diastrophic
dysplasia,
an abnormal
organization
of collagen
was found in areas
of interterritorial
matrix and around many degenerated
cells, but also in the lacunae
of cells without
ultrastructural signs of degeneration.
The segment-long-spacing
form of collagen
prepared
from cartilage
of three patients

with

diastrophic

dysplasia

showed

an

abnormal

cross-striation
pattern
in a portion
between
bands 42
and 45, corresponding
to the position
of the cx1(H) cyanogen-bromide-derived
10,5 peptide.
This suggested
that
in this syndrome
there is a structural
alteration
of the
type-Il
collagen
molecule.
There was an accumulation
of intracellular
lipid in
pyknodysostosis
and in hypochondrogenesis,
and of glyUnite

Malades,
address

VOL.

de Recherches

149,
reprint

66-A,

NO.

de G#{233}n#{233}tique
M#{233}dicale, H#{244}pitaldes

Rue de S#{232}vres, 75743

Paris
requests
to Dr. V. Stanescu.

6, JULY

1984

Cedex

IS,

France.

EnfantsPlease

the rationale

are

to primary

an abnormal
core protein
of a proteoglycan
species
is
not properly
transferred
to the Golgi system.
In Kniest syndrome,
intracytoplasmic
accumulation
of metachromatic
material,
dilatation
of rough endoplasmic
reticulum,
and an abnormal
gel-electrophoretic
pattern
of cartilage
proteoglycans
suggested
an abnormality
of cartilage
proteoglycan
metabolism.
Abnormalities
that probably
are related
to degradative
processes

M.D.*,

coproteins

search

strongly

STANESCU,

de Gen#{233}tiqueM#{233}dicale(INSERM).

is present
in the extracellular
matrix was not detected
by gel electrophoresis.
The accumulated
material
was
stained with antibodies
against the core protein of proThis

RITTA

Enfants-Malades.

ABSTRACT:
We performed
histochemical,
immunohistochemical,
electron-microscopic,
and microchemical studies
on cartilage
growth
plates from sixty-eight

patients

Incorporated

constitutional

abnormalities

development,
plate in these

for a classification,

or both.
disorders
aid

dis-

of cartilage
Studies
might

in accurate

or

of the
provide

diagnosis,

and therefore
permit
more
precise
prognosis
and genetic
counseling.
Studies
of this kind might also contribute
to our
understanding
of the normal
process
of endochondral
growth.
Finally,
the data obtained
from such studies
could
be useful
plasias

in establishing
the
syndromes
in humans
in animals.

We

have

had occasion

precise
relationship
between
and some osteochondrodys-

to study

the growth

cartilage

in

a relatively
large group
of normal
and osteochondrodysplastic
children.
The main aim of the study was to correlate
the histopathology,
growth
chemical

plate
data

the results
classification

histochemistry,

and ultrastructure

with
the microchemical
obtained
from the same
and their
of these

elsewhere394448.
that relate
to

the

significance
syndromes

In this paper
pathogenesis

of the

and immunohistospecimens.
Some

of

for the diagnosis

and
have
been
represented
we report
the main results
of several
osteochondro-

dysplasias.

Material
Case

and Methods

Material

Material

from

sixty-eight

children

with

nineteen

dif-

ferent
forms
of osteochondrodysplasias
was studied,
and
data on thirty-two
are presented
here (Table
I). The cases
were classified
according
to the international
nomenclature
of Paris8,

1976.

Cartilaginous

growth-plate

specimens

from
817

818

VICTOR

A newborn
child of thirty-six-weeks
the primary
trabeculae
are fine and
the late fetal and neonatal
periods.
are not disclosed
by microdissection

gestational
age who was apparently
normal (fresh-frozen
section,
Mallory
stain).
The columns
are regular
and
regular.
Two wide vascular
cartilage
canals
cross the plate.
(The system
of cartilage
canals
is well developed
in
Tissue
specimens
for biochemical
analysis
contain
appreciable
amounts
of non-cartilaginous
tissues
if the canals
on freeze-dried
sections.)

STANESCU,

RITTA

STANESCU,

FIG.

Fig. 2: Pseudoachondroplasia
irregular
arrangement
of cells
Fig. 3: Pseudoachondroplasia
of cells with large inclusions

AND

PIERRE

MAROTEAUX

in a seven-year-old
child
(Spurr
embedding,
one-micrometer
section,
azure
II-methylene
blue stain).
There
is an
many cells are large,
with intracytoplasmic
inclusions.
There
is metachromatic
material
around
the clusters
of cells.
in a five-year-old
child (Spun
embedding,
one-micrometer
section.
azure
Il-methylene
blue stain).
There
are groups
and eccentric
nuclei.
Some
nuclei
are pyknotic.
and

THE

JOURNAL

OF

BONE

AND

JOINT

SURGERY

PATHOQEN1C
TABLE

syndromet

Diastrophic

dysplasia

dysplasiat

Ages
(Yrs.)

5, 7.5,

6.5,

3, 6, 7

2.5,

Premature

Opsismodysplasi4

Newborn

Pyknodysostosist

14,

Hypochondrogenesisl

1 day

13

Spondyloepiphyseal
(atypical)t

dysplasia

2 (twins)

Spondyloepiphyseal
dysplasia
congenita
(atypical)t
*

In all patients

Biopsy
specimens.
Postmortem
specimens.

:t

the proximal

tibial

growth

newborn

14.5

older

orthopaedic
obtained

children
within

obtained
.

The

two to three

by

material
hours

after

growing

children

acute

with

apparently

Biopsy

Procedure

growth

The biopsies
plate were

cases,

a drill

Tissue

Preparation

normal

growth

(dead

from

dis-

of the proximal
tibial epiphyseal
performed
as described
before42.

biopsy

was

cartilage
In a few

used.

biopsy

fragments

were

divided

into

two

parts:

small one that was fixed for ultrastructural

study and a more
important
one that was quickly
frozen
with solid carbon
dioxide
and stored
at -40
degrees
Celsius.
Undecalcified
frozen
sections
were cut in a cryostat
sius. Groups
of forty-micrometer-thick

studied.

chemical

were

operations

normal

borns

determinations

sections
the

five

surgical
and new-

The

was

from

to 2 mos.

10 mos.

plate

obtained

10

5, 5, 6, 7, 7.5

Fibrochondrogenesist

were

(donors
of kidney
grafts
and patients
undergoing
repair
after accidents)
and from thirteen
fetuses
ease).

8, 9, 9

8, 8,

819

OSTEOCHONDRODYSPLASIAS

imens

No. of
Patients

Pseudoachondroplasiat
Kniest

IN

1*

Osteochondrodysplasia

Spondylometaphyseal
(Kozlowski
type)t

MECHANISMS

used

open

biopsy

from

newborns

during
was

death.

Control

spec-

that

were

seven

for histochemical

sections
was

were

were

and

at

staining.

with

The
the

perichondrium,

growth-plate

epiphyseal

,
,

..

of
to be

cartilage

and

thick,

forty-micrometer-thick

from

-...

groups

micrometers

and

bone,

alternated
four

freeze-dried

separated

20 degrees
Celsections
for micro-

#{149}s

.-

.r;-:

.-

.,,

iti.s
a

..

FIG.
Fig.

4: Pseudoachondroplasia
in a nine-year-old
blue. The bar indicates
ten micrometers.
Fig. 5: Pseudoachondroplasia
in a nine-year-old
metachromatic.
The bar indicates
ten micrometers.

VOL.

66-A,

NO. 6, JULY

1984

Fio.
child

(fresh-frozen

four-micrometer

section,

child

(fresh-frozen

four-micrometer

section,

Mallory
stained

stain).
with

azure

5
The

inclusions
A, pH

1 .75).

were
The

stained
inclusions

by aniline
are not

820

VICTOR

STANESCU,

FIG.

RITTA

STANESCU,

AND

PIERRE

MAROTEAUX

FIG.

Fig. 6: Pseudoachondroplasia
in a nine-year-old
child (ultra-thin
section.
Spun
embedding.
uranyl
acetate-lead
citrate
stain).
Large dilatations
of the
rough
endoplasmic
reticulum
with electron-lucent
and electron-dense
layers
can be seen. The bar indicates
one micrometer.
Fig. 7 (upper
right-hand
corner):
Gel electrophoresis
of dissociated
proteoglycans
(large-pore
polyacrylamide-agarose
gels).
1 = normal
child,
2 =
pseudoachondroplasia,
3 = diastrophic
dysplasia,
and 4 = mucolipidosis,
type III. A single
band is present
in pseudoachondroplasia.
Fig. 8: Pseudoachondroplasia
in a nine-year-old
child (fresh-frozen
four-micrometer
section).
This was not digested
by hyaluronidase
and was stained
with antibodies
against
the so-called
hook region
of the proteoglycan
protein
core (Fb fragments).
There
is positive
staining
of the inclusions.
The bar
indicates
ten micrometers.

cartilage
Light

proper

by microdissection,

as described

before49.

following

histochemical

procedures

were

applied

to the undecalcifled
frozen
sections:
azure A at pH 1 .75 and
pH 75,
Mallory
and van Gieson
stains34 after fixation
for
ten minutes
in 10 per cent formalin,
periodic
acid-Schiff
staining
after
twenty
minutes
of fixation
in 96 per cent
ethanol,
and von Kossa
staining34
after
ten minutes
of
fixation
in 4 per cent neutral
buffered
a-amylase,
and trypsin
digestions,
ing for tryptophan,
were performed
bedded

Sections
one micrometer
in Epon
812 or Spurr

azure

Il-methylene

blue

and

formalin.
Collagenase,
as well as Adams
stainas described
by Pearse28.

thick
from
fragments
medium
were stained
with

the

periodic

emwith

The

were

examined

with

Gel-Electrophoretic

with

Analysis

inhibitors26.

The

electrophoresis
gels2t .43,44

in 4 per

cent

101 Elmiskop

and
Ultracitrate

electron

mi-

of Proteoglycans

proteoglycans

on

and

Non-Collagenous

and

separated

The
fixed

tetroxide,
or both.
and lead

The microdissected
lyophilized
sections
four-molar
guanidinium
chloride35

change
chromatography
tion, two-molar
sodium

Microscopy

were

a Siemens

osmium
medium,
acetate

croscope.

Gel-Electrophoretic

fragments

cent

were

were
with

purified

extracted
protease
by

ion-ex-

on DE 52 in eight-molar
urea (fracchloride)43
and subjected
to gel

large-pore

polyacrylamide

agarose

acid-Schiff

technique.
Electron

in 2 per

post-fixed

embedded
in Epon
8 12 or Spurr
thin sections
stained
with uranyl

Microscopy

The

dehyde,

paraformal-

Analysis

of Link

Proteins

Proteins

proteins

extracted
from

with

four-molar

proteoglycans

by

THE JOURNAL

OF BONE

guanidinium

ion-exchange
AND

JOINT

chroSURGERY

PATHOGENIC

::

MECHANISMS

IN

#{149}N

C
.

.,

821

OSTEOCHONDRODYSPLASIAS

1,:

.%

#{149} --:

#{149}-b
#{149}%#{149}
A
...-.

.L!k

FIG.

FIG.

10

Fig. 9: Kniest
disease
in an eight-year-old
child (fresh-frozen
seven-micrometer
section.
von Kossa
stain).
There
is disorganized
columnization
with
a short proliferative
zone; rather
high cellularity:
large cells;
and short,
irregular
trabeculae.
Patchy
staining
with neutral
red is seen.
Fig. 10: Kniest
disease
in a ten-year-old
child (semi-thin
one-micrometer
section,
Spurr embedding,
azure Il-methylene
blue stain).
The basal cells
have large inclusions
with metachromatic
rims and extracted
centers.
Fibrillated
and non-homogeneous
zones are present
in the matrix.
The bar indicates
ten micrometers.

matography
chloride)

as just described
were reduced
and

(fraction,
analyzed

0.2-molar
by sodium

Immunohistochemical

sodium
dodecyl

On a limited

Methods

number

of specimens,

sulphate-polyacrylamide
gel electrophoresis4.
Bands
corresponding
to collagen
and to six major
non-collagenous
proteins
(two of them the link proteins)
are detected
by this

ical tests were performed.

were
a gift from
Dr.
France).
Immunolabeling

method.

by indirect
fresh-frozen

immunofluorescence
sections,
digested

decalcifled

with

Gel-Electrophoretic

The

Analysis

collagen

was

of the

solubilized

from

tilage by limited
pepsin
digestion22.
and analysis
by gel electrophoresis46
ready

the

of Collagen

extracted

car-

Further
purification2346
were performed
as al-

described.

Gel-Electrophoretic

Anal,vsis

of the

Cyanogen-Bromide-Derived

treated

a-Chains

Peptides

In some of the specimens,

with cyanogen
bromide

Major
from

the extracted
cartilage
was
according
to the method
of

Eyre and Muir9#{176},and the peptides

obtained
by sodium
dodecyl
sulphate-polyacrylamide
phoresis9#{176}46. Studies

that

type-I and type-Il

collagen
one was able to detect
collagen

0.46

VOL.

NO.

66-A,

6. JULY

1984

were

Collagen

performed

were analyzed
gel electroon mixtures

of

showed
that by using the method
to 10 per cent of the total type-I

and
IgG

a fluorescein
globulin5253.

EDTA

immunohistochem-

Antibodies
against
type-I
Grimaut
and Mr. Herbage
of type-I
collagen
was

using

on four-micrometer-thick
with hyaluronidase
the

purified

isothiocyanate-labeled
Antibodies
against

binding
region
of proteoglycan
against
chondroitin
sulphate
Heineg#{225}rd (Lund,
Sweden),

lgG

the

by antibodies
against
the Tamm-Horsfall
Dr. Heineg#{225}rd (Lund,
Sweden).
ofthe

For a limited
spacing
crystallites
from growth-cartilage

and

antibodies

sheep
anti-rabbit
hyaluronic-acid-

monomer
core protein
and
peptides,
a gift from Dr. D.
were
prepared
as has been

described5455.
The immunohistochemical
formed on four-micrometer-thick
fresh-frozen
the usual controls47.
An additional
control

Preparation

collagen
(Lyon,
revealed

Segment-Long-Spacing

tests

were
persections
with
was represented

protein,

Form

a gift from

of Collagen

number
of specimens,
segment-longwere prepared
from collagen
extracted
biopsy
specimens
. One-quarter-inch

822

VICTOR

STANESCU,

RITTA

STANESCU,

AND

PIERRE

MAROTEAUX

..

,,

..

k?

ak%*:

..\

..

.
.:,

.,
.

,t

-.

::;#{149}r#{149}4

--2..v

:A,

.-..

.i:-

--I-.

..-

..,

.4..-

.:

a.

..

..

I :.

#{149}-

...

..

.,

..

-1

,.

cL:mi
-

.,-,

..

.k._k%,ts.

-:

...#{149}

,c

-:-

?k

-..

..

#{182}

..-

.%-..
..-

..

..,,

..

0,.

FIG.

Kniest
disease
in a ten-year-old
child (Spurr
embedding.
endoplasmic
reticulum,
with fine fibrillogranular
material.
polyacrylamide-agarose
gels).
I = normal
and 2 = Kniest

sodium

by

washing

extensive

with

tube
against
several

and

the

solutions

0. 1 per
changes.

were

distilled

water.

One

to two-mu-

(0.4 to 0.8 milligram

per milliacid were placed
in the dialysis
dialyzed

cent acetic acid

The collagen

against
1 .5 milliliters
of freshly
osine triphosphate.
The knotted

for two
solution

11

ultra-thin
section.
uranyl
acetate-lead
citrate
stain).
Note
The bar indicates
one micrometer.
Inset:
Gel electrophoresis
disease.
Three
main bands are present
in Kniest
disease.

Visking
tubing
was prepared
by boiling
carbonate
for twenty
minutes
followed

limeter
collagen
solutions
liter) in 0. 1 per cent acetic

..p.

,-

#{149}).

(0.64-centimeter)
in 0.2-molar

at 4 degrees

Celsius

to three days, with

was then dialyzed

prepared
I per cent adenend of the dialysis
tube was

immersed
just
phate solution,
the

membrane.

degrees
grids,

below
giving
The

with

of

the surface
of the adenosine
only a small area for diffusion
whole

Celsius
for three
Drops of the solution
covered

the dilatations
of proteoglycans

procedure

was

to four days.
were applied

Formvar

films

carried

to 400-mesh
that

had

been

the rough
(large-pore

triphosacross
out

at 4

copper
lightly

coated
with carbon
by using an Edwards
evaporation
unit.
After ten minutes
the drops were removed
with filter paper
and the dried grids were stained
for ten minutes
with 1 per
cent phosphotungstic
acid, pH 3.4, rinsed briefly
with cold
THE JOURNAL

OF BONE

AND

JOINT

SURGERY

PATHOGENIC

MECHANISMS

IN

OSTEOCHONDRODYSPLASIAS

823

4,
FIG.

Spondylometaphyseal
pH 1 .75). The cell

has

distilled

dried,

cent
The

water,

dysplasia.
Kozlowski
large metachromatic

uranyl
acetate,
procedure
was

grids

were

stained
rinsed
carried

examined

type.
inclusions

for

again
out

ten

in a six-year-old
and narrow

minutes

with water,
at 4 degrees

in a Siemens

with

1 per

and air-dried.
Celsius.
The

101 Elmiskop

electron

microscope.

genita,
gested

that
volving

various
abnormalities
of cartilage
collagen,
lipids,
or glycoproteins.
spondyloepiphyseal
dysplasia
con-

a primary
defect
of cell division
by the presence
of a large number

chondrocytes.
In two

syndromes

the

study

was strongly
sugof multinucleated

revealed

an abnormality

is probably
closely
related
to the primary
proteoglycans
in pseudoachondroplasia

gen
in diastrophic
dysplasia).
morphological,
histochemical,
gation

pointed

proteoglycans,
however,
strongly

glycoproteins,

several
interrelated

complex
occur

defect
(inand colla-

In other
syndromes
the
and microchemical
inVesti-

to a predominant

zone) (fresh-frozen
lacunae.

to know

four-micrometer

whether

remote

effect

abnormality
lipids,

biological
in the growth

or cell

of collagen,
division.

As,

processes
that are
plate, it is difficult

primary

the primary
defect
abnormality
operating

opment,

as has

been

from

that we have described

Figure
1 illustrates
growth-plate

part

for

certain

animal

normal

of the tibia

Syndromes

with

or

in some
biodevelchon-

growth

the methods

were presented
in detail
the general
organization

elsewhere48.
of normal

in newborns.

The

gel-electrophoretic
normal
tibial
7 and the patpeptides
of

29.

Proteoglvcan

Abnormalities

Pseudoachondroplasia
In all of the five specimens
5), we found disorganized
proximal
tilage.
nests

In the proliferating
and clusters.
The

studied
tibial

(Figs.
2 through
growth-plate
car-

zone, the cells were arranged

hypertrophic
cells also showed

..

y
V
.

:
-,.

I
FIG.

Spondylometaphyseal
blue stain).
Many

VOL.

66-A,

NO.

dysplasia.
intracytoplasmic

6. JULY

1984

car-

with

cartilage

in Figure

A,

is a proximate

pattern
of proteoglycans
extracted
from
growth-plate
cartilage
is visualized
in Figure
tern of those
from cyanogen-bromide-derived
collagen,

azure

be a transitory
in early fetal

by studying

the proximal

with

In addition,

might
only

suggested

obtained

stained

trouble
defect.

syndromes
chemical

tilage

section.

the predominant
of the

drodysplasias83.
The results

Results
The studies
revealed
involving
proteoglycans,
In a patient
with atypical

12

child (basal
metachromatic

13

Kozlowski
type.
in a six-year-old
child (Spurr
vesicles
with metachromatic
granules
are present

embedding.
semi-thin
one-micrometer
in these cells of the basal zone. The

bar

section.
indicates

azure
Il-methylene
ten micrometers.

in
a

824

VICTOR

STANESCU.

RITTA

STANESCU,

FIG.

Spondylometaphyseal
dysplasia.
single,
smooth,
membrane-bound
bar indicates
one micrometer.

complete
instead
cessive
some
(Fig.
were

in a six-year-old
(lakes and spiral

loss of organization
into longitudinal
columns
and
formed
clusters
that contained
a few cells.
An examount
of matrix
separated
the clusters
of cells. In

very

large.

Some

cells

(Fig. 3).
The zone of provisional

were

pyknotic
was

and degenpresent

but

its width
was variable.
Vascular
invasion
was uneven.
In
some
areas
short tongues
of cartilage
penetrated
into the
metaphysis.
The primary
trabeculae
were rare, short, thick,
and irregular.
Some vascular
lacunae
were closed by transverse
bone-plates.
The osteoblasts
had no inclusions
and
rare osteoclasts
were found.
rial

The
(pH

histochemical
1 .75) disposed

tests showed
around
the

MAROTEAUX

14

Mallory

metachromatic
mateclusters
of cells.
The

stain

of the matrix

uranyl
with

was

showed
an uneven
zone
of
short,
irregular,
thick primary

proteoglycans.
blue

Staining

that the protein

with collagenase
the inclusions
was digested.

for

in the Mallory

acetate-lead
citrate
stain).
Large.
a bigger
granule
at one end. The

strong.

The

provisional
trabeculae.

The histochemical
study
showed
that the inclusions
were
and therefore
were not formed
(aniline

calcification

PIERRE

child (Spurr
embedding.
ultra-thin
section,
filaments
formed
by small granules,
often

places
the hypertrophy
of chondrocytes
was absent
2). Most of the cells in all of the zones of the cartilage
large and contained
inclusions
of various
sizes, some

of them
erated

Kozlowski
type,
vesicles
contain

AND

von

Kossa

stain

calcification

and

on fresh-frozen
sections
not metachromatic
(Fig. 5)
by glycosaminoglycans
or

proteins
procedure).

was

positive

Several

was probably
not procollagen.
followed
by Mallory
staining
were resistant,
whereas
Trypsin,
on the contrary,

(Fig.

tests

4)

showed

Digestion
showed
that

the cartilage
matrix
digested
the inclu-

sions, whereas
the staining
of matrix was almost
unchanged.
By using the procedure
of Adams
for tryptophan,
we found
that the inclusions
were strongly
positive,
and it is known
that procollagen
has a very low tryptophan
content.
The electron-microscopic
study showed
that the cells
THE

JOURNAL

OF BONE

AND

JOINT

SURGERY

PATHOGENIC

MECHANISMS

825

IN OSTEOCHONDRODYSPLASIAS

droitin

sulphate

tein

(Fig.

antisera

peptides

8).

They

against

of proteoglycan
were

only

link protein

and with

protein.
it is likely

that

the Tamm-Horsfall
Therefore,

monomer

very

control

reticulum

tration

of the

to the

extracellular

matrix

Kniest

prowith

antisera

against
an

is not processed
from the rough

Golgi

proteoglycan

core
stained

in pseudoachondroplasia

abnormal
proteoglycan
core protein
mally
and is not properly
transported
doplasmic

faintly

apparatus.

appears

The

noren-

concen-

to be decreased

in the

of the cartilage.

Disease

In all of the four

specimens

studied,

the columnization

of the cartilage
was disorganized
in an abnormally
proliferative
zone.
In the hypertrophic
zone,
irregular

short
col-

umns

basal

of several

enlarged

zone contained
large
seemed
hypercellular,

cells

were

cells with
especially

seen

(Fig.

9). The

cytoplasmic
in younger

inclusions
patients.

and
In all

of the zones of the growth

plate, scattered
foci of fibrillated
matrix
and of mucoid
degeneration
were found,
as well as
larger acellular
areas (Fig. 10). A Swiss-cheese
appearance
was not seen in Spurr-embedded
or fresh-frozen
sections
but was observed
as an

in paraffin-embedded

artefact

of processing

zone of provisional
trabeculae
were
teoblasts
The
stained
15

FIG.

Diastrophic
dysplasia
in a five-year-old
child
(columnar
area) (Spun
embedding.
one-micrometer
section.
azure Il-methylene
blue stain).
At a
higher
pH. metachromasia
is relatively
homogeneous.
Fibrotic
intercolumnar
septa are present
and many cells show various
signs of degeneration.
The hypertrophic
cells
are small
and often
degenerated.
with enlarged
multilayered
lacunae.

large vacuoles
bound
containing
a material

electron-dense

and

by the
with

electron-lucent

rough endoplasmic
alternatively
wide

layers

(Fig.

6). In some

abnormal

way

with

the

azure-A,

and periodic
acid-Schiff
methods
and with
semi-thin
sections,
the large intracytoplasmic
zone

rim (Fig. 10).

on fresh-frozen
sions

stained
The

The

seemed
unremarkable.
studies
showed
that the matrix

in a non-uniform

the basal

probably
matrix.

calcification
was present
but the primary
irregular,
short,
and rather
thin. The os-

and osteoclasts
histochemical

had

an extracted

center

Metachromatic
material
four-micrometer-thick
faintly
with the periodic
and did not stain with

lory methods
contained
reticulum,

sections33,

of the

electron-microscopic

dilatations
of the rough
cells, ofthe transitional

neutral
red.
inclusions

On
of

and a metachromatic
was seen
sections

acid-Schiff
Sudan
black

examination

in the cells
The incluand
B.

showed

Mallarge

endoplasmic
reticulum
and, in some
elements
between
this and the Golgi

cells,
the accumulation
was huge and the cells appeared
degenerated.
The inclusions
were similar
to those already
described
by several
authors532.
The collagen
fibers of the

system.
Their
doachondroplasia:

matrix

1 1).
an
was

were thin.
The gel-electrophoretic

glycans
The
mal
The

(Figs.

6, 7, and

gave

a single

amount

proteo-

an abnormal

pattern.

abnormal

(Fig.

7). The gel-electrophoretic

abnormal
proteoglycan
core
the matrix,
immunohistochemical
The inclusions
were strongly

protein

the

hyaluronic

region

VOL.

66-A,

1984

An excessive

of dissociated

proteins
gave a normal
pattern.
In order to see if the accumulated

NO. 6, JULY

background.

analysis

band

acid-binding

on an electron-lucent

8) disclosed

analysis
of a-chains
of collagen,
of the major
cyanogenbromide-derived
peptides
of collagen,
and of non-collagenous

was different
from that found in pseua fine fibrillogranular
material
was found

microtubules
seemed
to be present
in many cells (Fig.
The analysis
of dissociated
proteoglycans
showed

proteoglycans
extracted
from growth
cartilage
of norgrowing
children
yielded
two metachromatic
bands.
proteoglycans
extracted
from
pseudoachondroplastic

cartilage

content

was

Mallory,

protein

was

the

that was missing

from
studies
were performed.
stained with antibodies
against
and

against

the

chon-

evident
a-chains

pattern:

a third

abnormal

supplementary

band

of

(Fig.
1 1 , inset).
The gel-electrophoretic
analysis
of collagen
as well as that of cyanogen-bromide-

derived

peptides

normal

pattern.

and

of non-collagenous

proteins

showed

of
a

The relationship
between
the metachromatic
material
accumulated
in the rough
endoplasmic
reticulum,
the abnormal matrix,
the additional
population
of dissociated
proteoglycans,
and the keratan
sulphaturia
sometimes
found in
these

patients9

is still

to be elucidated.

Spondylometaphyseal

In the

three

Dysplasia,

specimens

studied,

Kozlowski

the

growth

Type

cartilage

826
contained

VICTOR

short

and slightly
or three

irregularcolumns.

groups

of two

fibrous
vascular

septa.
The hypertrophic
invasion
was regular

others.
Histochemical
1.75 in the columnar
chromatic
patchy

material

columns

staining
area.
was

in the intertemtorial

STANESCU,

separated

FIG.

wide

was present
and
areas and uneven

around

matrix.

The

the

cells

Mallory

AND

the
in

at pH
meta-

and

was

stain

was

PIERRE

MAROTEAUX

Electron-microscopic

areas

by

showed
metachromasia
In the basal
zone,
the

present

STANESCU,

In some

were
zone
in some

RITTA

of the chondrocytes
membrane.
They

study

showed

that

the inclusions

were large and were bound by a smooth

contained,
on an electron-lucent
back-

ground,
electron-opaque
material
that formed
granules,
flakes,
and spiral
filaments.
The latter appeared
as if they
were formed
by small granules,
often with a bigger
granule
at one end ofthe
A discontinuous

filament
suggesting
a proteoglycan
layer of electron-dense
material

17

FIG.

18

Fig.
16 (upper
left-hand
corner):
Diastrophic
dysplasia
in a seven-year-old
child (columnar
area) (fresh-frozen
seven-micrometer
stain).
The intercolumnar
septa are fibrotic
and there is abnormal
hypertrophy
of cells.
Fig. 17: Diastrophic
dysplasia
in a seven-year-old
child (zone of provisional
calcification)
(fresh-frozen
seven-micrometer
section.
There is regular
provisional
calcification.
Fig.
18: Diastrophic
dysplasia
in a six-year-old
child (basal
zone)
(Spun
embedding.
semi-thin
one-micrometer
section.
azure
stain).
The cells are degenerated
and the lacunae
are abnormal.
The bar indicates
ten micrometers.

strong
coarse

in the basal zone. The intercolumnar

septa contained
fibers.
Many cells from the basal and hypertrophic
zones contamed
large metachromatic
inclusions
(Fig.
12). On semithin sections
from Spurr-embedded
blocks,
the cells of these
areas contained
large intracytoplasmic
inclusions
with an extracted
center and a peripheral
metachromatic
rim (Fig. 13).
Von Kossa
staining
provisional
calcification,
mary trabeculae.
Some
bone.

revealed
a rather
regular
zone of
but short, thick, and irregular
prilacunae
were closed
by a bar of

quently
14).

found
The

along

the inner

formed
ditional

gel-electrophoretic
in two specimens,
rapid-moving
band

slower

than

phoretic
peptides
order

that

content.
was fre-

aspects

section,
von

Mallory

Kossa

stain).

Il-methylene

of the vacuoles

blue

(Fig.

study
of proteoglycans,
pershowed
the presence
of an adwith a migration
that was a little

of chondroitin

sulphate.

The

gel-electro-

analysis
of the major
cyanogen-bromide-derived
of collagen
gave a normal
pattern.

The data
involving

obtained
suggested
the proteoglycans,

a lysosomal
probably

type
limited

of disto the

chondrocytes.
THE

JOURNAL

OF BONE

AND

JOINT

SURGERY

PATHOGENIC

MECHANISMS

IN

827

OSTEOCHONDRODYSPLASIAS

Syndromes

with

Collagen

Abnormalities

Diastrophic

Dysplasia

The

cartilage

in six
short

growth

specimens,

was

but regular

columns,

of diastrophic
narrow,

dysplasia,

with

a narrow

and primary

studied
basal

trabeculae.

zone,

The

col-

umns were separated

by wide septa with a fibrillar
appearance.
In all zones
of the cartilage,
there were many cells
with condensed
or pyknotic
nuclei
in various
stages
of degeneration
(Fig.
15). Around
these cells, the lacunae
were
abnormally
matic

large

and

tonal
matrix
hypertrophic
were

FIG.

contained

They

(Figs.

concentric

zones

contained
cells were

pyknotic.

structure

and

metachromatic

had

15 and

(Fig.

zones
with
slightly
enlarged
large

lacunae

non-metachro18).

The

wide
and
with

interterri-

fibers.
The
some of them
a multilayered

18).

19

Diastrophic
dysplasia
in a six-year-old
child (Spun
embedding.
ultrathin section,
uranyl
acetate-lead
citrate
stain).
The collagen
is abnormally
organized
in the interterritorial
matrix
(sheets
with cross striations
in register.
wide fibers).
The bar indicates
one micrometer.

FIG.

21

Diastrophic
dysplasia
in a five-year-old
child (Spun
embedding.
thin section,
uranyl
acetate-lead
citrate
stain).
There arc thick fibers
cellular
lacunae.
The bar indicates
one micrometer.

The
showed

histochemical
that

an abnormal
of cells

upper

a strong,
uniform
stain in the basal

FIG.

Diastrophic
dysplasia
in a six-year-old
thin section,
uranyl
acetate-lead
citrate
lacunae
of an apparently
undegenerated
one micrometer.

VOL.

66-A,

NO.

6, JULY

1984

uniform
The

fresh-frozen

sections

at a low pH ( 1 .75)

It was positive

at the beginnings
ofthe
columns.

was more
unstained.

on

metachromasia

distribution.

situated
segments

staining
remained

study

azure-A

ultrain the

only

around

had

groups

of the columns
and in the
At pH 7, the metachromatic

and only the intercolumnar

periodic
acid-Schiff
method

septa
gave

stain. The Mallory

method
gave a strong
zone and in the dense fibers in the inter-

columnar
septa (Fig.
16). The von Kossa
stain showed
a
rather regular zone of provisional
calcification
(Fig.
17).
The ultrastructural
study showed
many degenerating
cells with one or several
concentric
halos containing
dense
bodies,
chondrocyte
remnants,
vesicles,
and wide collagen

20

child (Spun
stain).
Fibrils
chondrocyte.

embedding,
ultraare grouped
in the
The bar indicates

fibers.
sheets
abnormal

In many
areas
of the matrix,
with the periodicity
in register
periodicity

(Fig.

19). However,

the
and

collagen
formed
thick fibers with

some

chondrocytes

828

VICTOR

STANESCU,

RITTA

STANESCU,

FIG.

Segment-long-spacing
where

differences

of cartilage
in the

cross-striation

collagen
pattern

FIG.

23

from
are

a child

with

diastrophic

AND

PIERRE

MAROTEAUX

22

dysplasia

(a)

and

from

a normal

child

(b).

The

bar

indicates

the

region

present.

FIG.

25

Fig. 23: Fibrochondrogenesis

in a newborn
child (fresh-frozen
seven-micrometer
section,
Mallory
stain).
Numerous
network
in the epiphyseal
cartilage
proper.
Fig. 24 (upper
right-hand
corner):
Fibrochondrogenesis
in a newborn
child (fresh-frozen
seven-micrometer
section.
network
of connective
septa is positive
to the periodic
acid-Schiff
stain.
Fig. 25: Fibrochondrogenesis
in
a newborn
child (Spun
embedding.
one-micrometer
section.
azure
Il-methylene
surrounded
by nests of metachromatic
material
situated
between
fibrotic
septa.

THE

JOURNAL

connective
periodic
blue

stain).

OF BONE

septa

acid-Schiff
Elongated

AND

JOINT

form

a rich

stain).

The

cells

SURGERY

are

PATHOGENIC

FIG.
Fig. 26: Fibrochondrogenesis
metachromatic
material,
limited
Fig. 27: Fibrochondrogenesis

MECHANISMS

IN

OSTEOCHONDRODYSPLASIAS

829

26

FIG.

in a newborn
child
(fresh-frozen
by the connective
septa.
in a newborn
child (fresh-frozen

seven-micrometer
four-micrometer

section.
section,

Mallory

stain,
stain).

27
pH

Note

I .75).

Around

the elongated

=_L:__

3-

azure-A

the
cells

----

cells
and

are

nests

dense

of

septa.

1E1ZI

IL

1
FIG.

28

FIG.

29

Fig. 28: Fibrochondrogenesis

in a newborn
child (fresh-frozen
seven-micrometer
section).
Staining
with antibodies
to type-I
collagen
treatment
with hyaluronidase)
is positive
only around
the cartilage
canal.
Fig. 29: Sodium
dodecyl
sulphate-polyacrylamide
gel electrophoresis
of the major cyanogen-bromide-derived
peptides
of collagen
from
cartilage.
A, normal
newborn
child:
B, thanatophoric
dwarfism:
and C. fibrochondrogenesis.
The patterns
are similar
to one another.

VOL.

66-A,

NO.

6. JULY

1984

(after
growth-plate

prior

830

VICTOR

STANESCU,

FIG.

RITTA

the cell membrane

(Figs.
The gel-electrophoretic

AND

PIERRE

MAROTEAUX

30

FIG.

Fig. 30: Opsismodysplasia

in a newborn
child (fresh-frozen
vascular
penetration
is irregular.
Fig. 31 : Opsismodysplasia
in a newborn
child (fresh-frozen
with hyaluronidase).
There
is positive
staining
in the matrix

that did not show ultrastructural

many bundles
ofbanded
collagen
arranged
in the cell Iacunae
and

STANESCU,

seven-micrometer
seven-micrometer
of the abnormally

signs of degeneration
had
fibrils that were irregularly
often were in contact
with

20 and 21).
patterns
of proteoglycans.

non-

collagenous
proteins,
and cyanogen-bromide-derived
peptides of collagen
did not reveal
any abnormalities.
In a
severe case, a small quantity
oftype-I
collagen
was detected.
This seemed
to be related
to the intensity
and extension
of

section.

Mallory

section.
stained
wide hypertrophic

and

branched

31

stain).

There

is a wide

hypertrophic

to type-I

collagen

oval

nuclei.

with antibodies
zone.

(Fig.

25),

with

zone,
after

and

the

pre-treatment

However.

the

cytoplasm
was more dense and had more definite
limits than
the cytoplasm
of fibroblasts,
and around
the cells there were
nests of metachromatic
material
(Figs.
26 and 27). In the
growth
plate itself the septa were thinner
and the cells were
irregularly
disposed
and fusiform,
with
large
capsules.
Toward
the bone, the cells became
ovoid and larger and the
zones of provisional
calcification
and
the primary
trabeculae
were irregular.

vascular

invasion

and

the degeneration
of chondrocytes.
The cross-striation
patterns
of the segment-long-spacing form ofcollagen
prepared
from cartilage
ofthree
patients

The dissociated
proteoglycans
had a normal
gel-electrophoretic
fetal
pattern . The cyanogen-bromide-derived
peptides
of collagen
showed
the presence
oftype-lI
collagen

with diastrophic
prepared
from

and the absence

of type-I collagen.
ical study, the collagen
did not stain
I collagen.
The fibrosis
of cartilage

in a portion

dysplasia
were
normal
cartilage.
of the

crystallites

Bands
42 and 43 of the normal
a single,
very densely
stained
faintly
stained
compared
with
corresponded
to the position
rived 10,5 peptide
of human
cleavage-site

region

distinctly
different
The differences
between

bands

from that
occurred
42 and

45.

cartilage
were replaced
by
band.
Bands 44 and 45 were
normal
cartilage.
The region
of the cyanogen-bromide-dea(II)
and was close
to the

of collagenase

(Fig.

22).

On immunohistochemwith antibodies
to typein fibrochondrogenesis

seems
due, therefore,
to an abnormal
organization
of typeII collagen
(Figs. 28 and 29). The positive
staining
with the
periodic
acid-Schiff
method
(Fig. 24) as well as the normal
migration
of cyanogen-bromide-derived
peptides
argue
against
an underglycosylation
of collagen.
The underglycosylation
of collagen
in wide fibers3.

was

considered

as favoring

packing

Fibrochondrogenesis
In this lethal
chondrodysplasia4748.
the matrix
of the
epiphyseal
cartilage
was formed
by wide,
interwoven
connective
septa that were densely
stained
by the Mallory
and
van Gieson
methods
(Fig.
23). The cells were elongated

Opsismodvsplasia
In a child

with

dwarfism

feet and roentgenographic

yses, and the metaphyses,
THE

who

had very

short

hands

and

alterations
ofthe
spine. the epipha condition
that we named
opJOURNAL

OF BONE

AND JOINT

SURGERY

PATHOGENIC

FIG.

Pyknodysostosis
thin one-micrometer
dense
intracytoplasmic

MECHANISMS

IN

831

OSTEOCHONDRODYSPLASIAS

32

in a fourteen-year-old
child
(Epon
embedding.
semisection,
azure
Il-methylene
blue stain).
There
are
granules.
The bar indicates
ten micrometers.

FIG.

34

Atypical
spondyloepiphyseal
dysplasia
in a thirteen-year-old
embedding,
ultra-thin
section,
uranyl
acetate-lead
citrate
dilatations
of the rough
endoplasmic
reticulum
containing
material.
The bar indicates
one micrometer.

FIG.

Pyknodysostosis
in a fourteen-year-old
thin section,
uranyl
acetate-lead
citrate
inclusion
containing
lamellar
structures.
of an inclusion
into the Iacunae.
The

VOL.

66-A,

NO.

6,

JULY

1984

33

child (Spun
embedding.
ultrastain).
The chondrocyte
has a large
and the picture
suggests
expression
bar indicates
one micrometer.

FIG.

child (Spun
stain).
There
are
a fine granular

35

Atypical
spondyloepiphyseal
dysplasia
in a thirteen-year-old
child (Spun
embedding
semi-thin
one-micrometer
section,
azure
Il-methylene
blue
stain).
There
are chondrocytes
with several
nuclei,
and some of the nuclei
have blebs
and irregular
contours.
The bar indicates
ten micrometers.

832

VICTOR

STANESCU,

RITTA

STANESCU,

AND

TABLE
ABNORMALITIES
BASED

ON

PRESENT

CONVENTIONAL

IN

LIGHT
AND

THE

THE

CARTILAGINOUS

MICROSCOPY,

POSSIBLE

BIOCHEMICAL

IN
AND

SUGGESTED

HUMANS

WITH

CHONDRODYSPLASIA,

HISTOCHEMICAL
BY

THESE

AND

BIOCHEMICAL

STUDIES.

ABNORMALITIES

Abnormalities
Based
on Electron
Microscopy
and Histochemical
and
Biochemical
Studies

Abnormalities
Seen with
Conventional
Light Microscopy

Osteochondrodysplasia

PLATE

MICROSCOPY.

DEFECTS

MAROTEAUX

II

GROWTH

ELECTRON

PIERRE

Possible
Biochemical
Defect

Pseudoachondroplasia

Columns
replaced
by islands
of
cells; irregular
invasion
and
abnormal
primary
trabeculae:
large chondrocytes
with
intracytoplasmic
inclusions

Chondrocytes
contain
inclusions
with proteoglycan-monomer
core
protein
in rough
endoplasmic
reticulum:
gel electrophoresis
of
cartilage
proteoglycans
shows
absence
of a proteoglycan
population

Defective
processing
of core
protein;
defective
transport
of
core protein
from rough
endoplasmic
reticulum
to Golgi
apparatus

Kniest

syndrome

Irregular
columnization
and vascular
invasion;
cells with large
vacuoles,
especially
in basal
zone; matrix
patchily
stained,
with fibrillar
and mucoid
areas

Chondrocytes
metachromatic
large dilated
endoplasmic
fibrillogranular
electrophoresis
proteoglycan
metachromatic

Abnormal
synthesis
or processing
of
proteoglycans?
Relationship
with
keratansulphaturia
present
in
some patients?

Spondylometaphyseal
dysplasia,
Kozlowski
type

Short,
slightly
irregular
columns:
irregular
invasion:
short,
irregular
primary
trabeculae

Chondrocytes
of basal and
hypertrophic
areas contain
large
metachromatic
inclusions:
on
electron
microscopy
they contain
large vacuoles
bordered
by a
smooth
membrane
and containing
spiral filaments
formed
by
granules
suggestive
of
proteoglycans;
gel
electrophoresis
of cartilage
proteoglycans
shows
an
additional
rapid-moving
band

Disorder
of lysosomal
to chondrocytes?

Diastrophic

Narrow
basal zone: short,
regular
columns
separated
by wide
fibrotic
septa;
slightly
enlarged
and pyknotic
hypertrophic
cells:
many degenerated
cells; abnormal
lacunae:
zones of fibrotic
matrix

Abnormal
organization
of collagen:
wide fibers and tactoids;
wide collagen
bundles
in cell lacunae:
abnormal
distribution
of metachromasia
at low pH: abnormal
segment-long-spacing
pattern
of collagen

Abnormality
of structure
of type-Ilcollagen
molecule
(close
to
cleavage-site
region
of
collagenase)

dysplasia

sismodysplasia,

we found

hypertrophic
histochemical
lagen

in

analysis
pertrophic

an abnormally

wide

zone of the growth

cartilage.
studies
gave a strong
reaction
the

hypertrophic

area

of collagen
extracted
zone also disclosed

(Figs.

from
the

30

and irregular
The immunofor type-I col3 1 ). The

and

the microdissected
presence
of type-I

hycol-

lagen.
It is likely that the resorption
of cartilage
was impaired
due to the fact that the hypertrophic
cells secreted
predominantly type-I collagen
instead
of type-IT collagen
. In normal
growth-plate
cartilage,
type-I
collagen
is found
by immunohistochemical
cells

of the

Syndromes

methods
lower
with

Accumulation

only

hypertrophic

around

some

degenerated

zone.

Lipids

Pyknodvsostosis
The
tamed,

cartilage
in a patient
with
instead
of columns,
a narrow

of cells and had short,

The zone of provisional

pyknodysostosis
area of small

conislands

thick,
irregular
primary
trabeculae.
calcification
was narrow.
The car-

type

tilage was strongly

metachromatic
in the growth
around
the islets of cells and in the septa between
Ultrastructural

study

the presence

of abnormal

drocytes.

The

contained
mellar
bands

of the growth-plate
inclusions

inclusions

granular

were

material

structures.
The
with a periodicity

vacuoles
appeared

stained

with

Nile

and
ied.

The

33).

and

cartilage

irregularly

osteoblasts

revealed
of chonla-

many
Some

of them
pictures

into the cell lacunae.

by light microscopy

on frozen

sections.

characteristics
content
of the
and

and

interwoven

made up of dense parallel

to eight nanometers.
The

ofthe vacuoles
were visible
blue

and ultrastructural
phospholipid,

zone both
the islets.

in the majority

displaced
adjacent
structures,
and
to be related
to the Golgi apparatus.

chemical
probably

limited

single-membrane-bound

latter were
of seven

suggested
the expulsion
The abnormal
inclusions
and

an Abnormal
of Intracellular

contain
inclusions
and
areas of rough
reticulum
with fine
material;
gel
of cartilage
shows
an additional
band

The

histo-

indicated
a lipid,
vacuoles
(Figs.
32

the osteoclasts

were

not stud-

Hypochondrogenesis
The term achondrogenesis
osteochondrodysplasias
with
THE

JOURNAL

comprises
a lethal group of
severely
retarded
ossification
OF

BONE

AND

JOINT

SURGERY

PATHOGENIC

MECHANISMS

IN

TABLE
ABNORMALITIES
BASED

PRESENT

ON CONVENTIONAL

IN THE

LIGHT

AND

THE

POSSIBLE

II (Continued)

CARTILAGINOUS

MICROSCOPY,

GROWTH

ELECTRON

BIOCHEMICAL

PLATE

MICROSCOPY,

DEFECTS

IN HUMANS
AND

SUGGESTED

WITH

CHONDRODYSPLASIA,

HISTOCHEMICAL

BY THESE

AND

BIOCHEMICAL

STUDIES,

ABNORMALITIES

Abnormalities
Based
on Electron
Microscopy
and Histochemical
and
Biochemical
Studies

Abnormalities
Seen with
Conventional
Light Microscopy

Osteochondrodysplasia

833

OSTEOCHONDRODYSPLASIAS

Possible
Biochemical
Defect

Fibrochondrogenesis

Wide interwoven
connective
septa
in epiphyseal
cartilage
proper
and
basal zone,
with elongated
cells:
irregularly
disposed
fusiform
cells
and septa in growth
plate: ovoid
hypertrophic
cells;
irregular
provisional
calcification
and
vascular
invasion

Connective
septa formed
by type-Il
collagen;
around
the elongated
cells are nests of metachromatic
material:
normal
gelelectrophoretic
pattern
of
proteoglycans

Abnormal
collagen
cause

Opsismodysplasia

Abnormally
wide and irregular
hypertrophic
zone containing
wide connective
septa;
irregular
vascular
invasion

Type-I
collagen
hypertrophic

Abnormal
secretion
of type-I
collagen
by hypertrophic
chondrocytes
with impairment
cartilage
resorption?

Pyknodysostosis

Narrow
cartilage
with small
cells; short,
thick,
irregular
primary
trabeculae;
dark
inclusions
in many cells

islets

of

present
area

in abnormal

Single-smooth-membrane-bound
intracytoplasmic
inclusions
in
many chondrocytes
and in
cellular
capsules;
content
is
lamellar
(7-8-nm
periodicity):
Nile-blue-positive
inclusions
(frozen
sections)

organization
of type-lI
due to an unknown

Phospholipid
chondrocytes?
studied

disorder
of
Osteoblasts

Hypochondrogenesis

High cellularity:
reduced
matrix,
irregular
columns;
vascular
invasion
and trabeculae:
sclerotic
vascular
cartilage
canals

Many fat inclusions

in
chondrocytes:
gray,
amorphous,
not membrane-bound
inclusions
on electron
microscopy

Atypical
spondyloepiphyseal
dysplasia

Irregular
columns
with irregular
vascular
invasion
and primary
trabeculae;
large cells with
intracytoplasmic
vacuoles

Periodic-acid-Schiff-positive
inclusions
(after amylase
digestion)
stained
moderately
with aniline
blue: large dilatations
of rough endoplasmic
reticulum
with fine granular
material
on
electron
microscopy

Intracellular
glycoproteins

Atypical
spondyloepiphyseal
dysplasia
congenita

Ovoid
and
per
two
blebs

Normal

Defect
of cytokinesis
(due
membrane
or cell-skeleton
abnormality?)

and

abnormal

newborn
which

cartilage56.

children
we

with

propose

We
a minor

the

arrangement
of proliferating
hypertrophic
cells:
about 30
cent of chondrocytes
have
or several
nuclei,
often with
and irregular
contours

studied
form

designation

The children
were initially
genital
spondyloepiphyseal

material

from

to have
However,

percellularity

large

droblasts;
hypertrophy

irregular
ofcells;

wide,
irregular
chondrocytes
the cartilage
The

matrix

(fetal

feature);

column
arrangement
very irregular
vascular

primary
and little

trabeculae
endochondral

to those

of

chon-

with
evident
penetration
with

containing
degenerated
bone; and sclerosis

of

study,

performed

on

fresh-frozen

patients,
detected
in many chondrocytes
inclusions
that were positive
for Su-

dan B black. These

than those observed

inclusions
in the

pochondrogenesis

it was

VOL.

66-A,

examination

NO. 6, JULY

1984

were more frequent

and larger
controls.
In one child with hypossible
within

to perform
two
to three

The

inclusions

chondrocytes
that were
,

Syndrome

with

an electronhours
after

large

an

not

of

to

intracytoplasmic
and had a grayish,

content.

intracellular

of Glycoproteins

the cartilage
invasion

contained

accumulation

not membrane-bound

non-homogeneous

Accumulation

with
had

and

atypical

irregular

primary

spondyloepiphyseal
columns

with

trabeculae.

On

dysplasia,

irregular

vascular

histochemical

study,

the cartilage
showed
large
intracytoplasmic
inclusions
in
many chondrocytes.
The inclusions
were positive
to periodic
acid-Schiff
moderately
The

canals.
histochemical

picture

In a child
hy-

sections
from two
large intracytoplasmic

microscopic

death.
amorphous

severe
conthe histo-

relative

little

for

hypochondrogenesis48.

considered
dysplasia.

chemical
examination
revealed
lesions
similar
type-Il
achondrogenesis,
but less marked.
The main histopathological
features
were
with

seven

of achondrogenesis

histochemical

of

after a-amylase
digestion.
The inclusions
were
stained
with aniline blue by the Mallory
method.

electron-microscopic

rough

endoplasmic

terial

(Figs.

Syndrome

34 and
with

study
reticulum

showed

containing

distentions
a fine granular

of the
ma-

35).

a Primary

In monozygotic
twins
seal dysplasia2,
histological
were replaced
by an ovoid

impairment

of Cell

Division

with atypical
spondyloepiphystudy showed
that the columns
arrangement
of cells,
probably

834

VICTOR

STANESCU,

RITTA

STANESCU,

due to an abnormal
The histochemical

orientation
of the plane of cell division.
and microchemical
studies
did not reveal

evident

alterations

thin sections
drocytes
had

The

cytological

investigation

AND

lational
synthesis
plasmic

on semi-

charides;
linked

have been suggested:

in the rough
endoand with propeptides

at a later stage;
addition
of N-linked
oligosacand initiation
of the synthesis
of the 0-glycosideoligosaccharides.
The core protein
with its oligosac-

and irregular
contours.
These
findings
were confirmed
electron-microscopic
examination
of the chondrocytes.
Similar
findings
were
seen
in the cultures
of

skin

charides
in various
stages of maturation
must migrate
the rough endoplasmic
reticulum
to the Golgi region,

fibroblasts

sug-

the glycosaminoglycan

gested

and in leukocytes2.
an abnormality

The

in the

appearance

last phase

by

MAROTEAUX

4,24,36
Several
steps
of a core-protein
precursor
reticulum
with signal peptides

removed

showed
that about
30 per cent of the chontwo or several
nuclei.
Some nuclei
had blebs

PIERRE

strongly

of cell

Swarm-rat

division.

cursors4,

Discussion
The
complex
growth

pathogenesis

to a possible

previous
rarely,

defect

in proteoglycan

chemical

defect

normality
recently

In the

methods.

mechanism6394748,

sulphation,

due

primarily

study,

protein
animal

cartilage

ab-

chick2.

biopsy

the

An

synthesis
was
chondrodyspla-

the nanomelic

osteochondrodysplasias
microchemical
, and
syndromes,

to a defec-

has been described

a generalized
bioin cartilage31.

core
recessive

mouse2#{176}and

In several

most

Studies
of anadvanced.
A

probably

kinase,
represent

may

expressed

present

various
human
morphological,

defects
of the
for a few studies

the histopathology
or, more
or biochemistry,
or both, of the

of proteoglycan
suggested
in two

the cmdlcmd

primary
Except

syndromes29333742485657.
are somewhat
more

adenosylphosphosulphate
bmlbm
mice50,
and

sias:

osteochondrodysplasias,

pathogenic

studies
described
the histochemistry

cartilage
in various
imal chondrodysplasias

in

of human

group
of disorders
with
plate,
is poorly
understood.

pointing

tive

duces

study

of
using

growth
plates observed
by conventional
the abnormalities
demonstrated
by

scopic,

possible

histochemical,
biochemical

and
defect

biochemical
suggested

abnor-

light microselectron-micro-

studies;
and the
by their abnormali-

ties in several
of the chondrodysplasias.
In some
dysplasias,
the significance
of the abnormalities

chondrois not yet

clear.
For pseudoachondroplasia,
with an abnormal
accumulation
proteoglycan
population
in the
lum. The chondrocytes
contained
that,

clusions

inclusions
reticulum
zymatic

when

situated

huge,

seemed

in the

had responses
digestion
tests

very

the data were compatible

of the protein
core of a
rough
endoplasmic
reticularge intracytoplasmic
into produce
dilated

the

other

hand,

The

endoplasmic

to histochemical
staining
and enthat were compatible
with a non-

collagenous
protein.
They were positively
tibodies
against
a core protein
or proteins
On

cell death.

rough

a population

stained
with
of proteoglycans.

of proteoglycans

was

annot

detected
in the extract
ofcartilage.
Thus, it seems very likely
that an abnormal
core protein
is not properly
transferred
from the rough endoplasmic
reticulum
to the Golgi system.
The

biosynthesis

sis of a protein

core

of proteoglycans
or cores

and

their

requires
extensive

of the core
ably the
transported

-xyloside

Chondrocytes

of

treatment

pre-

of

chondrocytes

pro-

of

underglycosylated

a dominant

or the first stages

of a population

abnormal
protein
by the transport

of

core-protein

in pseudoachondroplasia

the synthesis

protein

added.

poo1

accumulation

that

alters

of processing

of proteoglycans

Prob-

is not properly
recognized
and
systems.
Other
mechanisms,

such as overflow
of the transport
system
due to hyperproduction
or alterations
of the transport
system
itself 27 seem
unlikely
since the trouble
is limited
to the core protein.
The proteoglycans
of growth
of baboons
can be separated
into

cartilage
two main

gel

of them

electrophoresis435.

extracts
suggests

that

the

or differ

processing
from

two

one

have

the early

of a common

core.

in the
strongly

This

protein

of post-translational

Similar

suggestions
and

degradation

and
by

is absent
different

steps

gel-electrophoretic25
but specific

of humans
populations

cartilage

populations

as regards

several

also

Only

of pseudoachondroplastic

studies354,

showed

malities
that revealed
the probable
pathogenic
mechanism
or offered
clues for further
research
on pathogenesis.
Table
II summarizes
the abnormalities
in the cartilaginous
copy;

mutation

are

contain

intracellular

protein36.
It is likely

cores

specimens

were studied
immunohistochemical

and
an

core

chains

sarcoma

from
where

resulted

immunochemical

of a common

core

was

suggested.

In diastrophic
dysplasia,
we found
an abnormal
organization
of type-I!
collagen
in many areas of the matrix
and in many chondrocyte
lacunae.
These
alterations
could
be related to the increased
number
of degenerated
cells found
in

this

syndrome.

found

around

throtic

articular

aging

human

Abnormal

degenerating
cartilage.
costal

collagen

organization

chondrocytes
Similar

cartilage7,

of normal

changes
and

were

prolonged

was

and

ar-

observed

in

prednisone

treatment
seemed
to enhance
these alterations6.
However,
in diastrophic
dysplasia
many
banded
collagen-fibril
bundles,

lular
tural

irregularly
arranged,
were visible
lacunae
of chondrocytes
that did
signs of degeneration.
The

abnormal

collagen

even in the pericelnot have ultrastruc-

organization

patients
with diastrophic
dysplasia
of an abnormality
in the structure

in the cartilage

of

might be the consequence

of the collagen
molecule,

as suggested
by the abnormal
segment-long-spacing
pattern
that we found in three specimens.
Indeed,
the banding
pattern of the collagen
molecules
organized
as segment-longspacing
crystallites
is considered
to be a molecular
fingerprint
ofthe
collagen
molecule,
showing
the distribution

the synthe-

of polar and apolar

amino
acids along
the molecule.
Our
findings
strongly
suggest
a genetic
defect
in the structure
or synthesis
of type-Il
collagen
in diastrophic
dysplasia.
Further
studies
on the cyanogen-bromide-derived
10,5 pep-

post-trans-

tide

of type-Il

collagen

molecule
THE

JOURNAL

and
OF

at the corresponding

BONE

AND

JOINT

SURGERY

PATHOGENIC
DNA

that

level could
is responsible

plasia.

The

perhaps
determine
the exact genetic
for the pathogenesis
of diastrophic

sensitivity

to mammalian

II collagen

in diastrophic

gated,

the

since

sections,

from

dyloepiphyseal
ilar

to

(stumpy

in

membranes

in

important,

the

in mice

mouse)7.
the

change
dysof type-

be also

investi-

to the cleavage

twins

congenita.

reported

IN

site

of the last phase of mitosis

was
studies
on semi-thin
and ultra-

monozygotic

produced

could

is close

region

dysplasia

those

dysplasia

collagenase

dysplasia

changed

of the enzyme.
A primary
impairment
demonstrated
by cytological
thin

MECHANISMS

role

last

phase

and a possible

the

of

atypical

findings

literature

by

The

with

The

for

recessive

the

of

cell

of

cell

is probably

ofthese

should

structures

be considered.
Thus,

the results

initial
various

disorder
origins.

several

possible

of the present

study

indicate

syndromes

mechanisms

we could

of growth

not obtain

a possible
pathogenic
specific
histological
The

existing

on normal

biochemical

data

and

still

the

be of
only

retardation

can

be

pathogenic
for further
For other

data

mechanism,
and
and histochemical

a pathogenic
classification
itary disorders,
but do
studies

that

in chondrodysplastic
cartilage
may
With a method
or group of methods,

examined.
With the method
used, the probable
mechanism
was revealed
or clues were obtained
research
on the pathogenesis
of a few syndromes.

found.

mutation
and

division

ofone

sim-

chondro-

stm

microfilaments

alteration

spon-

were

835

OSTEOCHONDRODYSPLASIAS

leading

only
some
alterations

do not provide

the

to
lesswere

basis

for

of this complex
group of heredgive some
indications
for further
abnormal

endochondral

growth.

References
1.

C. A. : AXELSSON,
INGE:
HEINEGARD,
DICK:
and GARDELL,
SVEN:
Extraction
and Purification
of Connective
Tissue.
Biochim.
Biophys.
Acta,
338:
108-119,
1974.
BATTIN.
J. : STANESCU,
V. : STANESCU,
R. ; MAROTEAUX,
P. ; and JOUSSEIN,
M.: Chondrodystrophie
secondaire
Chez deux jumeaux
monozygotiques.
Arch.
fran#{231}aisep#{233}diat. 34 (supplement
II): 233-239,
1977.
BRIGHTON,
C. T. : Structure
and Function
of the Growth
Plate.
Clin. Orthop.
, 136: 22-32,
1978.
CHAMINADE,
F. : STANESCU,
V. ; STANESCU,
R. ; and MAROTEAUX,
P. : Link-Proteins
and Non-Collagenous
dysplastic
Cartilages.
European
J. Pediat.
, 131:
237-245,
1979.
COOPER,
R. R. ; PONSETI,
I. V. : and MAYNARD,
J. A. : Pseudoachondroplastic
Dwarfism.
A Rough-Surfaced
Disorder.
J. Bone and Joint Surg. , 55-A:
475-484,
April
1973.
DEARDEN,
L. C. and MOSIER,
H. D. , JR. : The Effect of Prolonged
Prednisone
Treatment
on Human
Costal
ANTONOPOULOS,

2.

3.
4.

5.
6.

282,

1975.

of Proteoglycan

from

un trouble

Proteins

de Ia division

from

Normal

Endoplasmic
Cartilage.

Various

Am.

and

Reticulum
J. Pathol.

Types

cellulaire.

ChondroStorage

, 81: 267-

17.

L. C.: BONUCCI,
E.; and CUJCCHIO,
M.: An Investigation
of Ageing
in Human
Costal
Cartilage.
Cell Tissue
Res. , 152: 305-337,
1974.
J. : FAuRE, C. : GIEDION,
A. ; HALL, J. : KAUFMANN,
H. J. ; KOZLOWSKI,
K. : LANGER,
L. : LENZI, L. ; MAROTEAUX,
PIERRE:
MURPHY,
A.;
POZNANSKI,
A. K.; RIM0IN,
D.; SAUVEGRAIN,
J.: SILVERMAN,
F.; SPRANGER,
J.; STANESCU,
R.; and STANESCU,
V.: Nomenclature
des maladies
osseuses
constitutionnelles
International
Nomenclature
of Constitutional
Diseases
of Bone.
Ann. Radiol.
, 21: 253-258,
1978.
EYRE,
D. R. , and MUIR,
HELEN:
Characterisation
of the Major
CNBr-Derived
Peptides
of Porcine
Type
II Collagen.
Connect.
Tissue
Res. , 3:
165-170, 1975.
EYRE.
D. R. . and MUIR,
HELEN:
The Distribution
of Different
Molecular
Species
of Collagen
in Fibrous,
Elastic
and Hyaline
Cartilages
of the
Pig. Biochem.
J. , 151: 595-602,
1975.
GAY, STEFFEN,
and MILLER,
E. J. : Collagen
in the Physiopathology
and Pathology
of Connective
Tissue,
p. 39. New York,
Fischer,
1978.
GOETINCK,
P. F.: LEVER FISCHER,
P. L.: MCKEOWN-LONGO.
P. J.: QUINTNER,
M. I.: SAWYER,
L. M.: SPARKS, K. J.; and ARGRAVES,
W. S.:
Chondrogenesis
in Normal
and Mutant
Avian
Embryos.
In Levels
of Genetic
Control
in Development,
pp. 15-35.
Edited
by Stephen
Subtelny
and U. K. Abbott.
New York,
Liss,
1981.
GRYNPAS.
M. D.; EYRE, D. R.; and KIRSCHNER,
D. A.: Collagen
Type II Differs
from Type I in Native
Molecular
Packing.
Biochim.
Biophys.
Acta, 626: 346-355, 1980.
HASCALL,
V. C. , JR. , and KIMURA,
J. H. : Biosynthesis,
Secretion,
and Aggregation
of Proteoglycans
by Rat Chondrosarcoma
Chondrocytes.
Alabama
J. Med.
Sci.,
18: 29-35,
1981.
HEINEGARD,
D.; PAULSSON,
M.; and WIESLANDER,
J.: Heterogeneity
and Polydispersity
of Cartilage
Proteoglycans.
Sem.
Rheumatol.
, 11
(Supplement
I): 31-33,
1981.
HOLLISTER,
D. W.; BURGESON,
R. E.: and RIMOIN,
D. L.: Abnormal
Cartilage
Collagen
in Thanatophoric
Dwarfism
[abstracti.
Am. J. Hum.
Genet.
27: 783, 46A,
1975.
JOHNSON.
D. R.: The Growth
of Femur
and Tibia in Three Genetically
Distinct
Chondrodystrophic
Mutants
of the House
Mouse.
J. Anat. , 125:

18.

JOHNSON,

19.

G.:
BRILL, PAULA;
RAAB,
F. ; HIRSCHHORN,
K. : and MATALON,
R.: Kniest
Syndrome
with Dominant
Inheritance
and
Am. J. Hum.
Genet. , 27: 755-764,
1975.
KIMATA,
K0JI;
BARRACH,
H.-J.;
BROWN,
K. S. ; and PENNYPACKER,
J. P. : Absence
of Proteoglycan
Core Protein
in Cartilage
from the cmd/cmd
(Cartilage
Matrix
Deficiency)
Mouse.
J. Biol. Chem.,
256: 6961-6968,
1981.
MCDEVITT,
C. A. , and MUIR, HELEN: Gel Electrophoresis
of Proteoglycans
and Glycosaminoglycans
on Large-Pore
Composite
PolyacrylamideAgarose
Gels.
Analyt.
Biochem.,
44: 612-622,
1971.
MILLER,
E. J.: Structural
Studies on Cartilage
Collagen
Employing
Limited
Cleavage
and Solubilization
with Pepsin.
Biochemistry,
11: 49034909, 1972.
MILLER.
E. J. : MARTIN,
G. R. ; PIEZ,
K. A. : and POWERS,
M. J. : Characterization
of Chick Bone Collagen
and Compositional
Changes
Associated
with Maturation.
J. Biol. Chem.
, 242:
5481-5489,
1967.
MUIR,
I. H. M. : The Chemistry
of the Ground
Substance
of Joint Cartilage.
In The Joints
and Synovial
Fluid,
edited
by Leon Sokoloff.
Vol. 2,
pp. 27-94.
New York,
Academic
Press,
1980.
OEGEMA,
T. R. , JR. ; BROWN,
MARTIN;
and
DZIEWIATKOWSKI,
D. D. : The Link Protein
in Proteoglycan
Aggregates
from the Swarm
Rat
Chondrosarcoma.
J. Biol. Chem. , 252: 6470-6477,
1977.
OEGEMA,
T. R. : HASCALL,
V. C. ; and DZIEWIATKOWSKI,
D. D. : Isolation
and Characterization
of Proteoglycans
from the Swarm
Rat Chondrosarcoma. J. Biol. Chem.
250: 6151-6159,
1975.
PALADE,
GEORGE:
Intracellular
Aspects
of the Process
of Protein
Synthesis.
Science,
189: 347-358,
1975.
PEARSE,
A. G. E. : Histochemistry.
Ed. 3, vols.
1 and 2. London,
Churchill,
1968.
PEDRINI-MILLE,
ANGIOLA,
and PEDRINI,
VITT0RI0:
Proteoglycans
and Glycosaminoglycans
of Human
Achondroplastic
Cartilage.
J. Bone and
Joint Surg.
64-A:
39-46,
Jan. 1982.
PENNYPACKER.
J. P. : KIMATA,
K0JI;
and BROWN,
K. S.: Brachymorphic
Mice (bmlbm):
A Generalized
Biochemical
Defect
Expressed
Primarily
in
Cartilage. Devel. Biol., 81: 280-287, 1981.
PERLE,
M. A., and NEWMAN,
S. A.: Talpid2
Mutant
of the Chicken
with Perturbed
Cartilage
Development
Has an Altered
Precartilage-Specific
Chromatin
Protein.
Proc.
Nat. Acad.
Sci.
77: 4828-4830,
1980.

7.

DEARDEN.

8.

DORST,

9.
10.
1 1.
12.

13.
14.
15.

16.

267-275,

D. R. , and
31: 319-328, 1974.
KIM,
H. J.; BERATIS,
Mucopolysacchariduria.

20.
21

1978.

22.
23.
24.

25.
26.
27.
28.

29.
30.
31.

VOL.

HUNT,

D. M.: Biochemical

N.

66-A,

NO.

6.

JULY

1984

Observations

on the Cartilage

of Achondroplastic

(can)

Mice.

J. Embryol.

and

Exper.

Morphol.,

836

VICTOR

32.
33.

37.
38.
39.

44.

45.

46.
47.
48.
49.
50.
51.
52.

TIMPL,

RUPERT;

WICK,

VON

DER

GEORG;

J. Immunol.

MARK,

HELGA;

WIESLANDER,

Biochem.

55.
56.
57.

WIESLANDER,

JORGEN,

J.

179:

and

35-45,

JORGEN,

and

Meth.

VON

Embryo
by Immunofluorescence.
Chick
Embryo.
Devel.
Biol.
54.

PIERRE

MAROTEAUX

S. J. : A Rough Endoplasmic
Reticulum
Storage
Disease
in Chondrocytes
[abstract].
Anat.
Rec. , 166: 363,
1970.
RIM0IN,
D. L.; SILBERBERG,
RUTH:
and HOLLISTER,
D. W.: Chondro-Osseous
Pathology
in the Chondrodystrophies.
Clin.
Orthop.,
114: 137152, 1976.
ROMEIS,
BENNO:
Mikorskopische
Technik.
Munich,
Oldenbourg,
1948.
SAJDERA,
S. W. , and HASCALL,
V. C. : Proteinpolysaccharide
Complex
from Bovine
Nasal Cartilage.
A Comparison
of Low and High Shear
Extraction
Procedures.
J. Biol. Chem.
244: 77-87,
1969.
SCHWARTZ,
N. B.: Biosynthesis
of Chondroitin
Sulfate
Proteoglycan.
In Lysosomes
and Lysosomal
Storage
Diseases,
pp. 63-72.
Edited
by J. W.
Callahan
and J. A. Lowden.
New York,
Raven
Press,
1981.
SILLENCE,
D. 0.; HORTON,
W. A.: and RIMOIN,
D. L.: Morphologic
Studies
in the Skeletal
Dysplasias.
A Review.
Am. J. Pathol.,
96: 8 13-870,
1979.
SPURR, A. R.: A Low-Viscosity
Epoxy
Resin Embedding
Medium
for Electron
Microscopy.
J. Ultrastruct.
Res.,
26: 31-43,
1969.
STANESCU,
RITTA;
STANESCU,
VICTOR:
and MAROTEAUX,
PIERRE:
Abnormal
Pattern
of Segment
Long
Spacing
(SLS)
Cartilage
Collagen
in
Diastrophic
Dysplasia.
Collagen
and Rel. Res.
2: 1 1 1-1 16, 1982.
STANESCU,
V. , and MAROTEAUX,
P. : Gel Electrophoretic
Studies on Proteoglycans
and Collagen
ofAbnormal
Human Growth
Cartilage:
Proteoglycan
Abnormalities
in Pseudoachondroplasia
and in Kniests
Disease.
Pediat.
Res. , 9: 779-782,
1975.
STANESCU,
VICTOR,
and STANESCU,
R. : Morphological
and Biochemical
Studies
in Growth
Cartilage
of Osteochondrodysplasias.
In Klinische
Genetik
in der padiatrie,
pp. 31-53.
Edited
by J. Spranger
and M. Tolksdorf.
New York,
Thieme,
1980.
STANESCU
, V. ; BONA,
C. ; and IONESCU,
V. : The Tibial
Growing
Cartilage
Biopsy
in the Study of Growth
Disturbances.
Acta Endocrinol.
, 64:
577-601,
1970.
STANESCU,
V. ; MAROTEAUX,
P. ; and SOBCZAK,
E. : Gel Electrophoresis
of the Proteoglycans
of the Growth
and of the Articular
Cartilage
from
Various
Species.
Biomedicine,
19: 460-463,
1973.
STANESCU,
VICTOR:
MAROTEAUX,
PIERRE:
and SOBCZAK,
ELIANE:
Proteoglycan
Populations
of Baboon
(Papio-papio)
Articular
Cartilage.
GelElectrophoretic
Analysis
of Fractions
Obtained
by Density-Gradient
Centrifugation
and by Sequential
Extraction.
Biochem.
J., 163:
103-109,
1977.
STANESCU,
VICTOR;
MAROTEAUX,
PIERRE: and SOBCZAK.
ELIANE:
Proteoglycan
Populations
of Baboon
(papio
papio)
Cartilages
from Different
Anatomical
Sites.
Gel Electrophoretic
Analysis
of Dissociated
Proteoglycans
and of Fractions
Obtained
by Density
Gradient
Centrifugation.
Biochim.
Biophys.
Acta,
629: 371-381,
1980.
STANESCU,
V.; MAROTEAUX,
P.; and STANESCU,
R. : Etude par #{233}lectrophor#{232}se
sur gel, des chaines
a et des CNBr
peptides
du collagene
du cartilage
du croissance
dans les chondrodysplasies.
Ann. genet. , 19: 1 19-125,
1976.
STANESCU,
V.: MAROTEAUX.
P.: and STANESCU,
R.: The Biochemical
Defect
of Pseudoachondroplasia.
European
J. Pediat.,
138: 221-225,
1982.
STANESCU,
VICTOR;
STANESCU,
RITTA:
and MAROTEAUX,
PIERRE:
Etude
morphologique
et biochimique
du cartilage
de croissance
dans
les
ost#{233}ochondrodysplasies.
Arch.
fran#{231}aisep#{233}diat., 34 (supplement
I): 1-80,
1977.
STANESCU,
V. ; STANESCU,
R. : and SZIRMAI,
J. A. : Microchemical
Analysis
of the Human
Tibial Growth
Cartilage
in Various
Forms of Dwarfism.
Acta Endocrinol.
, 69:
659-688,
1972.
SUGAHARA,
KAZUYUKI,
and SCHWARTZ,
N. B. : Defect
in 3phosphoadenosine
Sphosphosulfate
Formation
in Brachymorphic
Mice.
Proc. Nat.
Acad. Sci. , 76: 6615-6618,
1979.
SZIRMAI,
J. A.: Quantitative
Approaches
in the Histochemistry
of Mucopolysaccharides.
J. Histochem.
and Cytochem.,
11: 24-34,
1963.
Immunohistology.

53.

AND

40.

43.

STANESCU,

36.

42.

RITTA

PHILLIPS,

34.
35.

41

STANESCU,

DER

GAY,
,

MARK.

18:

STEFFEN:

165-182,
KLAUS:

Antibodies

and

GAY,

I. Preparation
of Collagen
48: 237-249,
1976.

HEINEGARD,

DICK:

to

Distinct

Types

of

Collagens

and

Procollagens

and

Their

Application

in

of the

Chick
of the

1977.

Immunochemical

STEFFEN:

Type

I and
Analysis

Study

of

Type
of

Differential

II Specific
Cartilage

Collagen

Synthesis

Antibodies

and

Proteoglycans.

Their

during

Development

Application

Antigenic

to Early

Determinants

of

Stages

Substructures.

1979.
and

HEINEGARD.

DICK:

Immunochemical

Analysis

of Cartilage

Proteoglycans.

Radioimmunoassay

of the

Molecules

Substructures.
Biochem.
J. , 187: 687-694,
1980.
YANG, S-S.;
BROUGH,
A. J.: GAREWAL,
G. S.; and BERNSTEIN,
JAY:
Two Types
of Heritable
Lethal
Achondrogenesis.
J. Pediat.,
85:
1974.
YANG,
S-S.;
HEIDELBERGER,
K. P.: BROUGH,
A. J.: CORBETT.
D. P.: and BERNSTEIN.
JAY: Lethal
Short-Limbed
Chondrodysplasia
Infancy.
in Perspectives
in Pediatric
Pathology.
edited
by H. S. Rosenberg
and R. P. Bolande.
Vol. 3, pp. 1-40. Chicago,
Year Book
1976.

THE

JOURNAL

OF BONE

AND

JOINT

and

the

796-801,
in Early
Medical,

SURGERY