STUDIES
ON
BONE
K. LITTLE
MATRIX
M.
and
The
purpose
of
bones.
decreased
in width
formation.
Two
osteoporosis
that
the
paper
In
osteoporosis,
elderly
will
types
are
disuse
is not
part
be
considered
OSTEOPOROTIC
A. COURTS,
LONDON,
BONE
ENGLAND
centre,
Oxford,
and the
Association,
Holloway,
London
attention
bone
on
the
atrophy,
compact
the
bone
and
normal
ageing
component
1962) so
the
matrix
of
normal,
trabeculae,
is thin.
osteoporosis
of the
mineral
and Buhr
Holdoway
AND
and
focus
or
the
the
of
to
and
common
(Little,
OXFORD,
is
number,
composition
changes
matrix
this
and
in the
KELLY,
NORMAL
Fro,n the Nuffield
Orthopaedic
Gelatine
and Glue Research
British
osteoporotic
IN
Resorption
and
are
exceeds
osteoporosis
process.
old
histologically,
It has
new
in the
elderly.
already
been
is not affected
by ageing
structure
and composition
or
bone
This
shown
pathological
of the bone
here.
MATERIAL
Control
specimens-Pieces
chemical
analysis,
bone
(taken
taken
from
41-50,
at necropsy)
subjects
51-60,
over
60
also
Osteoporosis.
following
81 and
either
unfixed
taken
from
the
femur.
groups:
Most
cortical
a series
only
femoral
vertebral
was
used
for
Fresh
were
2, 2-10,
taken
1 1-20, 21-30,
from the under
31-40,
2 and
were
specimens
from
bodies
taken
fresh
at
specimens
end
necropsy,
and
fixed
specimens
was
for
in rabbits’
humerus
giraffe.
from the bison
and the phalanx
from Dremotherium-a
The human
specimens
came
from
a British
cemetery
bison
of Allier
was
from
in France,
the
and
the
last
ice age,
was
about
material
specimens
using
for
the
chemical
method
analysis,
microscopy.
bones-Available
1 50. The
in cases
chemical
this
and
two
only.
tibiae,
used
from
material
bone.
of
of fracture,
group;
three
the fixed
to cortical
microscopy
were
each
least
Fragments
in cases
specimens
at
or the vertebrae
of the femur.
For
used.
electron
produced
electron
were
femur
used
Fresh
for
specimens
of the
were
disuse
osteoporosis
and Trueta
(1958).
were
from
with
60.
From
in addition
the femora
of the neck
from
taken
femora,
over
femur,
of fracture
necropsy
were
fixed
Ancient
A.D.
bone
microscopy.
three
specimens
under
formalin
taken
or as a result
shafts
Rabbit-Experimental
described
by Geiser
deposits
Cortical
for electron
and at least
specimens
bone
of
were
at necropsy
prepared
for electron
microscopy.
Biopsy
specimens
from the upper
both
age
over.
Human-Specimens
diagnosed
and
from
each
specimens
from
taken
and cancellous
bone
for chemical
analysis,
age group,
and more
from
subjects
of cancellous
bone were taken
from each
in
analysis
were
of the
71-80,
microscopy,
were
subjects
bone
groups.
electron
specimens
were
in each
61-70,
age
For
of
both
cortical
was used
and
humerus
and
the
and
vertebrae
from
Dremotherium
30,000,000
years
the
forerunner
at York
from
was
from
human,
of the deer
approximately
the
the
and
Oligocene
old.
METHODS
Electron
the only
microscope
microscopy-Fixed
and unfixed
specimens
of bone were examined.
Formalin
was
fixative
used,
because
with
necropsy
material
the more
commonly
used
electron
fixative,
osmium
tetroxide,
instead
of fixing,
actively
degrades
cells;
and there
is
also
danger
of
the
that
phosphate.
in a refrigerator
calcium
either
soil
was
44 B,
VOL.
D
shaken
NO.
precipitated
or washed
3,
AUGUST
particles
of heavy
The specimens
prepared
or in a fat solvent
such
out
1962
as far
metal
might
without
fixation
as trichlorethylene.
be misinterpreted
were
With
stored
the
as crystals
until
required
ancient
bones,
as possible.
503
504
K.
In
uncalcified
cell processes
decalcified.
bone
the
LITTLE,
mineral
passing
through
They
were
then
methacrylate,
and
after
KELLY
masks
the
AND
most
the canaliculi
washed
and
sectioning
All the electron
microscope
times.
This relatively
low
M.
of
the
matrix
can be clearly
embedded
in
embedding
photographs
magnification
A. COURTS
structure
medium
was
removed
of tissue sections
shown
allows
easy comparison
component
substance,
The bundles
of collagen
Hydrochloric
complete
the
After
of
bone
are closely
treated
soluble
residue.
The
and
with
adherent,
(Courts
of the
various
as
the
diffraction
defatting
alkali
of the collagen,
has been called
eucollagen
remainder
chosen
x-ray
decalcification
were
was
characteristic
characteristics
modified
form
The
acid
then
1959,
1960);
was
left
fractions
obtained
in Figure
and
pattern
acetate.
were
of
citric
tissue
examined
and
When
which
the
we have
fat dissolved
decalcification
was
disappeared.
degradation
did not
This
was used.
The pieces
destroy
the molecular
the solubility
in acid buffers.
dissolved
in buffers
has been
be reprecipitated
with
;< 4,000.)
4) collagen,
hydroxyapatite
acid.
(
removed,
agent.
enhanced
the fraction
it may
behind,
soft
2
of the matrix.
Canaliculi
are
is dry, so that there are spaces
of matrix
in decalcifled
bone.
FIG.
are present.
2; and
of limited
with
but considerably
eucollagen
and
collagen
and canaliculi
decalcifying
a process
and
amyl
2,000 to 5,000
examined
with
on which
the mineral
is laid down);
3) the second
which
is seen to glue together
the individual
collagen
fibrils,
and also the bundles
of fibrils,
as shown
concentrated
on in this work.
The bone was cut into pieces,
the marrow
out.
although
therefore
isobutyl
object
on a black background.
components:
1) the mineral,
component
of the ground
FIG.
1
1-Section
of undecalcified
bone.
The mineral
masks
the fine structure
and cell processes
from osteocytes
pass through
them.
The section
where
fluid would
be present
in life.
( x 10,000.)
Figure
2-Section
Figure
present,
(black)
1),
with
are magnified
with sections
the light microscope.
All the photographs
shown
will be ofa white
Chemical-Bone
contains
a number
of readily
distinguishable
which
can be removed
by acid;
2) a polysaccharide-containing
substance
(it is probably
this
main component
of the ground
(Fig.
seen.
Specimens
were
a mixture
of butyl
and
some
of the
by x-ray
THE
JOURNAL
in a pure
ground
form
This
termed
by dialysis.
substance,
as
a solid
diffraction.
OF
BONE
AND
JOINT
SURGERY
STUDIES
Details
was
bone
of the
freed
from
and
The
time
for
mineral
MATRIX
experimental
marrow
and
were
removed
three
days
for
necessary
NORMAL
are
cut
into
to change
was
the
The
Fresh
pieces
post-mortem
femoral
sections.
bone
were
shaft
Cancellous
then
soaked
in
also acted as a preservative.
treatment
in 8 per cent
weight/volume
To complete
decalcification
in this
by
temperature.
hydrochloric
of
505
BONE
transverse
The
fat.
removed
at room
OSTEOPOROTIC
as follows.
adhering
bone
days
AND
one-inch-long
mechanically.
to dissolve
of the
seven
IN
procedure
tissue
portion
acid
it was
BONE
adhering
trichlorethylene
hydrochloric
ON
acid
solvent
daily.
X-ray
diffraction
photographs
FIG.
3
Diagram
to illustrate
main reflections
on x-ray diffraction
photographs.
A-The
reflection
with spacing
286 A is characteristic
of collagen
; it represents
a spacing
along
the axis of the collagen
fibre.
B-This
reflection
represents
a spacing
between
collagen
molecules,
and varies with the humidity.
C-This
reflection
is
weak in dry collagen,
and strong
in photographs
of wet collagen.
D-A
halo,
caused
by diffraction
of x-rays from non-crystalline
regions
of polymeric
compounds.
E-This
sharp reflection
gives the spacing
of the most intense
ring
of the saturated
fatty acid powder
pattern.
The sharpness
indicates
a material
of
larger crystallitesize than the collagen.
F and G-Two
prominent
reflections
in the soluble
eucollagen
pattern.
H and I-The
corresponding
reflections
seen
in patterns
from specimens
containing
the tubular
form of collagen.
J-A
strong
reflection
observed
in the residue,
after chemical
treatment,
from the matrix
of
osteoporotic
bone.
were
taken
then
freed
hours
in
hydroxide
of the
from
20
VOL.
in
44 B,
cent
was
Centigrade
The
per
for
sodium
treated
water
3,
AUGUST
that
sulphate
days,
were
overnight.
1962
decalcification
overnight
to
the
by
solution,
and
washed
with
The
three
collagenous
the
was
allowing
The
time
complete.
water.
which
after
specimen.
which
was
in running
100 millilitres),
added
to ten
pieces
running
NO.
per
again
four
to ascertain
by washing
(8 grammes
Centigrade,
then
bone
acid
mixture
bone
had
changes
material
of
The
It was
then
used
it to cool
was
taken
to
dissolve
at
an
sodium
20 degrees
at
a jelly-like
was
twenty-four
to about
sodium
then
collagen
for
maintained
on
saturated
was
bone
soaked
21
degrees
appearance.
chloride,
approximately
and
506
K.
neutral
pH.
Extractions
for forty-eight
paper
(Whatman
precipitated
were
hours.
No.
The
540)
soluble
LITTLE,
M.
made
into
solutions
and the
collagen
was
in the
present
work
it was
FIG.
O1
were
filtrate
AND
molar
used
at
A.
COURTS
citric
filtered,
dialysed
centrifuged
protein
concentration
from about
1 per
over
phosphorus
pentoxide.
X-ray
precipitated
soluble
collagen,
and the
X-ray diffraction-X-ray
diffraction
is
but
KELLY
first
for
acid
8,000/G
for
cent to 5 per cent, and
diffraction
photographs
dried
insoluble
remains
most
solely
commonly
When
material,
atoms
weak
usually
of being
a beam
the
in a plane
reflection.
have
sharp,
of
x-ray
FIG.
monochromatic
their
will
molecules
be
orientated
suitably
set
the
collimated,
of
planes
reflection,
and
of compounds
ordered.
In
In such
non-crystalline
cases
regions
and from these regions
are obtained
crystal
gives a series
of spots:
a well
the reflected
x-rays
fall on a flat plate.
collagen.
The
collagen
temperature
contains
small
THE
through
water.
filter
The
increase
the
to
of structure
the
analysis,
components.
5
FIG.
7
Figure
5-Unorientated
from an osteoporotic
bone.
collagen.
collagen
each
in a strong
(a group
imperfectly
broadened.
amount
of ordering,
A well ordered
single
gives a series
of arcs when
from
from
will usually
result
Polymeric
compounds
limited
obtained
x-rays,
is diffracted
minutes
as a method
4
6
patterns.
Figure
4-Orientated
6-Wet
collagen.
Figure 7-Residual
beam
thirty
of characterising
FIG.
X-ray
diffraction
collagen.
Figure
at room
then dried in a vacuum
desiccator
were
then
taken
of the dried
of the matrix.
used
as a method
(pH22)
through
gauze,
and then
three days against
running
JOURNAL
impinges
of atoms.
on
a crystalline
Densely
packed
sparsely
packed
atoms
in a
to which
collagen
belongs)
the
x-ray
of polymers
reflections,
there
instead
is often
a
very diffuse
reflections.
ordered
and orientated
fibre
Figure
4 shows
the pattern
crystals,
OF
or crystallites,
BONE
AND
JOINT
and
SURGERY
STUDIES
when
these
Figure
5
In
are
ON
in a disordered
x-ray
main
categories.
There
Such
are
differences
differences
for
the
of
deciding
patterns
variations
type
will
smaller
is so
into
507
BONE
rings,
in
the
caused
and
the
pattern
shown
in
is a collagen.
6 are
of
not
and
SPACINGS
OF
SHOWN
IN
A.
286
THE
a broad
the
superimposed
on
halo
bone
the
of larger
collagen
crystallite
size
rH
472
3.94
hand,
regions
in
the
structure
in a pure
state,
but
mixed
polysaccharide-containing
463
position
D (Fig.
subjects,
had
a
This
collagen.
The
saturated
other
+0.05
pattern.
than
in the
B, on the
a
criteria
400
11.
oferror
in the
diffraction
of compounds
reflection
G.
elderly
best
4.55
J.
is also
of the
has
PATTERNS
IF.
to 36
of
some
which
ANGSTROMS
428
which
matrix
3 IN
limits
displace
3,
I
FIGURE
Probable
possibility
crystallites,
When
collagen
is in contact
prominent,
and
diffraction
present
DIFFRACTION
337
53
E.
give
reflection
crystalline
to 107
C.
D.
may
three
3.
being
poorly
is the
Figure
as one
Disordered
D in Figure
collagen
Non-crystalline
B. 143
from
The
obtained.
position
in
regarded
into
condition.
larger
water
A
according
to the humidity.
the reflection
C becomes
Figure
There
from
adsorbed
be
a polymer,
fall
in physical
5.
4 and
reflection
of
variations
reflections
Also
it might
specimens
differences
Figures
the
TABLE
components
by
by
that
of
these
in sharper
collagen
specimen
possibility
materials.
number
collagen
crystallites.
approximate
THE
main
out
With
result
constant
a given
shown
in the
is always
other
material
OSTEOPOROTIC
a large
as exemplified
which
mammalian
or not
halo
There
obtained
AND
spread
of
the
from
In
Angstrom,
the
a broad
with
are
noticed.
first
size,
atoms.
whether
of
give
arcs
taken
minor
variations
in spacing
water
or an aqueous
solution
with
NORMAL
usually
in orientation,
of 286
shows
are
reflections
planes
spacing
are
in crystallite
broadened
the
IN
photographs
in pattern
of
array
diffraction
variations
and
MATRIX
obtained.
is
minor
of
BONE
fatty
3).
Some
sharper
indicates
spacing
acid
diffraction
patterns,
reflection
the
presence
corresponds
series-myristic,
E
(Fig.
of
another
3)
with
that
of
palmitic
and
stearic
the
acids.
There
is
also
structure.
The
crystalline
regions
manner.
the
possibility
molecules
There
are
these
could
may
be
that
collagen
composed
not
a situation
of
itself
a number
analogous
not
of
be arranged
always
is
of
constant
different
in similar
to that
of the
composition
amino
acids,
proportions,
nylons,
or
which
are
and
and
in
the
in a similar
also
polymers,
and in which
there
are a number
of different
compounds,
each being
a polyamide,
but with
minor
variations
in structure
and,
therefore,
in the x-ray
diffraction
pattern
(Little
1959).
When
the diffraction
patterns
of collagens
from various
sources
are examined
such differences
are
found.
soluble
In collagen
eucollagen,
from
the
primitive
diffraction
anlage
patterns
and
the
show
growth
cartilage,
a consistent
reflections
F and G differ from those of H and I (Fig. 3) obtained
from tendon,
or when
it is obtained
from
the residual
collagen
described
above.
A modified
diffraction
pattern,
with
the
(Fig. 3), is given by the residual
collagen
suffering
from osteoporosis
(Fig. 7).
VOL.
44 B,
NO.
3,
AUGUST
1962
when
the
bone
as well
sample
as reprecipitated
when
after
strong
the
The spacings
of the
diffraction
pattern
is
the
chemical
has
been
difference.
reflection
taken
separation
in
from
position
patients
J
508
K.
LITTLE,
M.
KELLY
AND
COURTS
A.
Experimentally,
the radiation
one-quarter
of a millimetre
thick;
of samples
the specimen
to film
interplaner
spacings
collagen
measurement,
since
obscured.
In
examined,
with
mixtures
I values
with
types
an
but
the
for
of photographs
Comparison
both
that
were
Table
measurements
for
was
specimens
used was copper
Kcx; the
a Porter
type fibre camera
distance
was 3 centimetres.
described
by Astbury
(1943).
using
matrix,
8 and
of the
caught
added
can
also
been
with
be shown
286
value
same
FIBRILS
articular
reflections
Angstroms
for
suitable
D (Fig.
given,
as an internal
the
spacing
of
for
3) might
are
be
from
standard.
this
reflection
open
by
ends
the
about
on
the
640
and
the
IN
cartilage
THE
and
ELECTRON
other
MICROSCOPE
connective
tissues,
from
slit.
specimens
Angstrom
can
be shown
two main
types
of collagen
fibril.
One is thick,
of
and threadlike.
The two varieties
are present
in both
the
form
photographs,
of fibrils
knife
9
of the lack of ground
substance-the
a normal
matrix.
In suitably
oblique
to have
in stereoscopic
information
in discussions
various
the
were
halo
taken
of the edge of a section,
where
much
of the ground
substance
shadowed
to increase
contrast.
Coarse
collagen
fibrils
can be seen,
fibrils.
(x 20,000.)
Figure 9-Section
showing
osteoid
matrix
from a
formation
of ground
substance.
Both
types
of collagen
fibril
can be
cell processes
passing
into canaliculi.
The dark
spaces
are caused
by
loss of fluid.
(x 6,000.)
COLLAGEN
than
some
the
reflection
gives
microscope
to contain
and the other
is thin
fibrils
be shown
OF
9, in which-because
if there
was
clearly
collagen
of
patterns
of the
FIG.
in common
electron
diameter,
Figures
diffraction
vicinity
8
APPEARANCE
more
the
the
of collagen.
FIG.
by the
varying
all
in the
spacings
standard
Figure
8-Electron
microscope
photograph
is missing.
The section
has been
metal
mixed
with
very fine and
more
flexible
specimen
in which
there
was defective
seen.
Also present
is an osteocyte
with
Bone
the
made
external
not
lines
specimens
were approximately
was used.
For the majority
The
method
for obtaining
Films
from
over 400 separate
of hollow
of which
tendon
This
method
(Little
bands.
an
have
been
of
tubes
(Kennedy
example
cut
1958).
It is these
is seen
THE
in
while
thick
after
other
JOURNAL
OF
more
BONE
that
this
fibrils
are
drastic
AND
be seen
thicker
10.
frequently
fibrils
the
and
Figure
stereoscopically
Banding
can
the
1955),
is shown
obliquely,
viewing
fibrils
sections
JOINT
can
Here
have
gives
illustrated
chemical
SURGERY
STUDIES
treatment
This
for
results
the
in
the
ON
BONE
isolation
of
removal
of
MATRIX
IN
reticulin
the
NORMAL
from
material
some
of
the
photograph
tubes
have
been
banding
FIG.
Figure
1 I-Transmission
and
reticulin
involved
almost
shadowless.
because
treatment
Section
of epiphysial
has
lost,
is present
so
that
on both
with
within
and
fibrils
collagen.
a number
the inside
photograph
and
In the processing
of
fibrils
removed
the
ground
cartilage.
shown.
substance
through
which
1952).
collapse
and
outside.
the contents
seen
that
it can
be
(x 10,000.)
reticulin
12
membrane
from
fairly drastic
alkali treatment,
and the collagen
fibrils appear
The section
has been metal shadowed
at an angle of 1 in 5.
growth
Kramer
which
10
FIG.
microscope
509
BONE
(Little
the
II
electron
of the
of fibrils of tubular
OSTEOPOROTIC
specimens
contained
FIG.
Stereoscopic
AND
normally
surrounds
Part of the vertically
orientated
collagen
Only fine fibrilsare present.
(x 6,000.)
kidney.
Isolation
as collapsed
Banding
ribbons,
is prominent
(x 50,000.)
Figure
12between
columns
of cells is
fibrils.
appear
as ribbons.
Shadows,
which
would
have
been
cast
by round
fibrils,
are lacking,
and banding
is now seen very clearly
(Fig.
11). It can also be seen on some of the fibrils
in
Figures
8 and 10, because
chemical
treatment
has removed
some
of the ground
substance
VOL.
44 B,
NO.
3,
AUGUST
1962
510
K.
with
which
the
by either
collagen
alkali,
a longitudinal
inside
and
The
fine
substance
the
640
and
while
fibrils
an
A.
COURTS
Banding
which
been
never
is rarely
is normally
obtained
the
the
seen
used
without
in some
banding
to
the
form
predominantly.
types
12)
of fibril
to
can
have
it is most
fibrils
(Fig.
two
observed
prominent,
cartilage
tubular
of these
been
is less
tenacity
growth
one
covered.
has
have
increased
contains
only
AND
or trypsin,
spacing
epiphysial
tendon
containing
usually
of a fibril
Angstrom
showing
cartilage
KELLY
be
treatment
other
seen
centres.
to
be
both
tube.
thread-like
the
are
M.
specimens,
section
outside
although
fibrils
as in these
When
LITTLE,
at
this
contain
spacing.
the
X-ray
show
the
this
easily
fine
tubular
by
Tissues
such
fibrils
diffraction
form,
detected
and,
ground
as anlage
almost
patterns
appropriate
the
exclusively,
of specimens
pattern.
#{149}
‘L
p.
‘5
#{149},‘.
S
.J,-
-.L
I.
‘‘v
p\
5..
.5..
a
(?.
#{149}
‘‘S
-.
‘-
‘5’
*
.-
k.
‘J
-.
-
--
11C’e.
a.’,.
.‘,
#{149}
11
:‘,.
.S.,
..
FIG.
Figure
13-Section
processes
remain
from
13
F1G.
formalin
in the canaliculi.
fixed
infant
femur,
(x 6,000.)
Figure
containing
shrunken
NORMAL
decalcified
with
HUMAN
number
from
and
of subjects
the
femoral
chemical
were
shaft
analysis
in the
was
were
age
used,
groups
but
both
cent
ninety-eight
over
cortical
hydrochloric
bone
acid.
matrix,
with
Cell
lacunae
BONE
The bones
examined
included
specimens
from still-born
from subjects
in each decade
; the eldest subjects
from whom
microscopy
8 per
14-A
section
of decalcifled
osteocytes.
(x 6,000.)
14
and
sixty.
For
and
and infant
cadavers,
specimens
were taken
eighty-seven
respectively.
the
analysis
chemical
cancellous
bone
was
and several
for electron
A large
cortical
used
for
bone
electron
microscopy.
Normal
bone is shown
fully calcified
in Figure
1, and fixed and acid
both
specimens
being
from
a middle-aged
subject,
but the appearance
age to another.
Fixed
and decalcified
bone from the femur
of an infant
decalcified
in Figure
2,
varies
little from
one
is shown
in Figure
13,
in which
relationship
sections
often
canaliculi.
the cell processes
in the canaliculi
have remained
of each bundle
of fibrils
to the osteocyte
14 to 18 show
marked,
both
other
examples.
in the calcified
The
(Fig.
intact.
These
and its associated
differences
in the density
of the
17) and decalcified
(Fig.
16) state.
THE
JOURNAL
show the
Figures
trabeculae
are often
very
This implies
that more is
OF
BONE
AND
JOINT
SURGERY
STUDIES
Figure
15-A
similar
section
with each osteocyte,
through
Figure
16-Section
of fixed
between
bundles
of collagen
ON
BONE
MATRIX
IN
NORMAL
The
(x
density
NO.
3,
AUGUST
BONE
511
3,000.)
Ft..
round
the osteocyte
is greater
than in the portion
of
Figure 18-Section
of fixed and decalcified
cortical
bone.
(x3,500.)
44 B,
OSTEOPOROTIC
to Figure
14, but cut in a different
plane.
e numerous
canaliculi
associated
which
cell processes
can pass through
the matrix,
can be seen.
(x 6,000.)
and decalcified
cancellous
bone.
In the trabeculae
the difference
in density
is frequently
more marked
than in the compact
bone illustrated
in Figures
2,
13 and 17. (x3,500.)
.3.17
Figure
17-Section
of calcified
trabecula.
bone seen at the side of the photograph.
VOL.
AND
1962
512
K.
LITTLE,
M.
KELLY
AND
A.
COURTS
S.,.
.,
.‘
.,f. 1’,.
_#{149};_SI,
-
I
Figure
bundles
Section
I
;
FIG.
19
FIG.
20
19-Section
of unfixed bone which has been decalcified
with 8 per cent hydrochloric
acid. The collagen
appear
almost
unaffected
by the lack of fixation,
and are still closely adherent.
(x 4,000.)
Figure 20cut from reprecipitated
soluble
eucollagen.
Fibrils
are fine and randomly
arranged
in a felt-like
mass,
with no noticeable
banding.
(x 10,000.)
FIG.
21
FIG.
Figure 21-A
chondrocyte
in primitive
anlage cartilage.
The surrounding
matrix,
containing
no tubular
fibrils,
is more compact,
but otherwise
has a similar
appearance
to the precipitated
eucollagen.
The area of parallel
bands of endoplasmic
reticulum
which can be seen in the cytoplasm
of the cell is believed
to be responsible
for the formation of the matrix. (x 5,000.)
Figure
22-Section
from specimen
of bone, from eighty-sevenyear-old subject after defatting, decalcification,treatment with caustic soda, and then citric acid. Dense material
has accumulated
in some canaliculi.
(x 6,000.)
THE
JOURNAL
OF
BONE
AND
JOINT
SURGERY
STUDIES
involved
than
matrix,
as
When
but
the
simple
unfixed
there
is rather
compared
with
the
dispersed
years
old.
The
When
appearance
of the
pattern
diffraction
longer
communication
of
shown
that
bone
areas
treatment
that
boiling
the
the
the
account
last
given
I 50
A.D.
of the
were
hydrochloric
so
clearly
swelling
also
seen
of
The
as
the
clear
The
Isleworth,
that
in decalcified
fresh
is
(Fig.
19)
chemical
type
shown
last
the
architecture
the
electron
to the
gave
elderly
acids.
X-ray
centimetres,
in
be due
to the
Cohen
allow
preliminary
greater
(1962)
cells.
to
the
and
ease
known
has
that
that
after
fatty
it
alkali
diffraction
pattern
subject
acid,
citric
of
others
It is probable
x-ray
the
subjects
(Fig. 22)
a dense
acid.
FOSSILS
of
normal
Some
in Figure
was
described,
human
decalcified,
23).
glaciation
bones
normal
bones
unfixed,
preserved,
substance
the
the
fatty
75
fibrils
tubular
normally
saturated
on the
the
in
of
be liberated
though
Vigorous
separation
of the ground
showed
well
of
above
might
diffraction
of fine
be compared
eighty-seven-year-old
soda
and
with
was
(Fig.
21).
of living
cautiously
bone
(Fig.
it is already
found.
the
can
of
matrixup to two
X-ray
suggested
shows
entire
infants
dispersion
some
(1962),
matrix
bone
at
from
also
this
may
AND
were
of
ofthe
of a bison
been
which
the
of collagen
Attempts
Middlesex)
of
These
the component
radius
BONES
architecture
andthediffractionpatternwas
it was
has
reticulin
acid,
which
appearance
types
matrix.
It
be devoid
the
from
appearance
free
distance
Sissons
In
myristic
canaliculi,
examined.
acid.
can
of
film
In the specimen
from an
treatment
with
caustic
two
specimens
which
from
to
that
by
occurs.
free
the
intact,
processing
Dispersion
described
specimens
1959)
areas
difference
in some
section
the
adults.
the
cells
processing
Work
OLD
In
uniform
remains
of
unfixed
a felt-like
a mixture
Courts
age.
by
cartilage
a specimen
large
release
accumulated
otherwise
suggest.
pattern.
occasionally,
observed.
and
and Windrum
1954).
defatting,
decalcification,
material
513
architecture
and
fibrillar
gave
only
of
with
quite
will
an
remainder
of the
produced
anlage
presence
old
is
occasionally
better
in
the
children
the
seen
after
Little
matrix
in old
is in these
(Little
after
the
general
matrix
eucollagen
were
5, but
be
(Kelly,
hydrolysis
of
might
the
older
gelatin
collagen
the
in
showed
primitive
taken
to
calcification
BONE
part
of the collagen-almost
in those
specimens
taken
less
around
Figure
spacings
much
soluble
were
OSTEOPOROTIC
When
fibrous
a large
eucollagen
forms
indicating
patterns
of
decalcified,
in the
whereas
the
of
amount
destroyed.
fractions
Other
of
AND
specimens.
was
matrix
pattern
a
fixed
separated
20).
residue
diffraction
the
the
NORMAL
in a microradiograph
are
partly
reprecipitated,
(Fig.
the
cohesion
eucollagen,
microscope
gave
more
proportion
soluble
The
and
IN
seen
of bone
shrunken
of these
fibrils.
in
commonly
chemical
experiments
in citric acid as soluble
photographs
produces
MATRIX
difference
specimens
are
In
BONE
appearance
cells
similar,
when
a
the
ON
showed
with
the
in Great
after
8 per
cent
were
not
decalcification
resulted
no
eucollagen,
remaining
Britain
(Willments
acid
decalcification
an
circa
fibrils
5. From
the microscopic
which
acts as a glue was
preserved
and
from
in
appearance
also present.
Gravel
(Fig.
Pit,
24).
and
the matrix
again
gave an excellent
diffraction
pattern
of tubular
collagen.
The 30,000,000
years old phalanx
of an animal
called
Dremotherium,
from the Oligocene
deposits
at Allier
in France,
was obtained,
of which
a radiograph
is shown
in Figure
25. The
architecture
of this bone did not remain
intact
on decalcification,
the ground
substance
holding
bundles
found.
of fibrils
together
The appearance
was
intact
still
(Fig.
25).
dissolving
in the acid,
of these bundles
varied,
It is clear that collagen
so that separated
bundles
of collagen
were
but in many
the tubular
variety
of collagen
is a very stable
compound.
OSTEOPOROSIS
The
affected
VOL.
specimens
and
44 B,
NO.
of
diagnosed
3,
AUGUST
osteoporotic
at
1962
necropsy,
bone
or
were
from
taken
biopsy
either
specimens
from
elderly
taken
subjects
from
patients
severely
with
514
K. LITTLE,
M.
KELLY
AND
A.
COURTS
FIG.
23
Section of demineralised human
bone from approximately
AD.
150.
Decalcified
with
8 per cent
hydrochloric
acid.
The architecture
of the bone
is preserved,
and cell lacunae
can be distinguished.
Decalcification
in this specimen
has resulted
in a slight swelling
of the
matrix.
(x 5,000.)
Figure
24-Section
of demineralised
bone (bison)
from the last glaciation.
Decalcifled
with 5 per cent
hydrochloric
acid.
The architecture
of the bone is again preserved,
with the bundles
of collagen
remaining
closely
adherent.
Details
in this photograph
may be compared
with Figure 2. (x 10,000.)
Figure
25Section of Dremotherium
bone after demineralisation
with 5 per cent hydrochloric
acid.
Collagen
bundles
remain
intact (after about
30,000,000
years)
but the ground
substance
holding
the bundles
together
has
dissolved in the acid.
(x 6,000.)
Inset-Radiograph
of phalanx
of Dremotherium,
from the Oligocene
deposits
of Allier.
THE
JOURNAL
OF
BONE
AND
JOINT
SURGERY
STUDIES
fractured
necks
fibrillar
to
The
alkali,
and
normally
This
suggests
result
( 1958)
served
the
individual
one
on
of
bone,
examined.
by any
operation
decalcification
in
the
bundles
of
of the
structure
lost
It
collagen.
removal
pattern
pattern
collagen
of the
in the
fixation,
microscope.
its
was
not
26).
degradation
diffraction
without
electron
(Fig.
515
BONE
the
was
et a!.
that
of the
material
shown
1959)
with
of some
reprecipitated
residue
(Kelly
Treatment
in solution
was
in Figure
which
might
7.
be
the
reaction.
also
osteoporosis
the
OSTEOPOROTIC
mass
resulted
diffraction
variant
of rabbit
were
so that
AND
after
showed
extraction,
The
5.
of a chemical
Samples
of collagen
acid
collagen,
a third
matrix,
separation
by citric
NORMAL
a structureless
boundaries
in Figure
IN
bone
as
of chemical
insoluble
shown
MATRIX
The
appeared
distinguish
results
followed
that
BONE
of femur.
form,
possible
ON
made
osteoporotic
The
most
bundles
osteocyte
patients
disuse
by the
method
observation
of collagen
apparently
with
by disuse
significant
were
acting
seen
was
of Geiser
that
in
to degenerate,
as a unit
osteoporosis
was
(Figs.
found
show
the
28).
the
and
early
with
27 and
to
the
matrix
Bone
same
Trueta
stages
of
being
obtained
at
characteristics.
DISCUSSION
Previous
work
pathological
(Little,
changes
Holdoway
affected
here
to the organic
matrix,
and
pathological
states.
in which
polysaccharide-containing
Buhr
of
changes
have
matrix
which
is
the
not
the
shown
to occur
there
fundamental
collagen
commonly
ageing
nor
has
in certain
many
been
given
physiological
substance-predominantly
of which
the
of
neither
Attention
ground
alter
form
most
that
mineral.
been
collagen,
did
form
showed
bone
contains
component-and
of the matrix
In osteoporosis
and
1962)
composition
Organic
Chemical
modification
x-ray
fibre
diffraction.
modification,
and
the
are
normally
two
structure,
which
is less
described,
a
types.
as shown
by
soluble
after
undergoes
degradation
changes.
It is necessary
ageing
bone
will
When
also
die,
bones
has
shown
intact
for
millions
In the
with
when
are
to consider
process.
dry,
since
associated
matrix
in the
changes
from
and
absence
in the
the
collagen
body,
or when
The
examination
mineral.
of destructive
could
occur
death
of
agents,
collagen
saturated
by,
in any
occurs,
the
ancient
in the
normal
cells
and
matrix
in the
fossilised
can
remain
of years.
the
or an
disrupted.
if these
is removed
leaving
that,
body
water
first
bone
matrix
is in contact
aqueous
the changes
These
ground
solution
in the
statements
with,
do not
substance,
and
it would
diffraction
since
tissue
fluid.
When
saturated
be expected
to be in a more
reactive
state than
pattern
suggest
that some of the hydrogen
bonds
refer
to pure
the evidence
collagen,
available
but
concerns
to collagen
collagen
and
which
the closely
has
not
been
completely
separated
from it. For example,
the diffraction
pattern
shown
in Figure
5 and in the
others
like it were obtained
from human
tissues which still had ground
substance
present.
In work
such as that of Bowes,
Moss
and Raistrick
(1960),
who have investigated
the degradation
of
collagen
substance.
collagen
holding
seen
was
by
irradiation,
specimens
also consisted
of collagen
fibrils
held together
by ground
When
animal
skin was irradiated
dry, using
50 megarad
gamma-irradiation,
the
appeared
largely
unaffected,
but the ground
substance
was obviously
degraded,
no longer
the bundles
ofcollagen
fibrils together.
The final result (Fig. 29) was analogous
to that
in the
fossil
completely
structureless,
has
fibrils
taken
some
44B,
When
comparable
place.
To
sections
hydroxide
(Fig. 30).
The second
point
occur
when osteocytes
VOL.
25).
(Fig.
different.
NO.
3,
AUGUST
fibrils
with
the
the
see
of
effect
tendon
to consider
die, thereby
1962
the
No
same
were
matrix
of this
were
irradiation
seen,
the
in advanced
type
treated
dose
bundles
was
given
coalesced
osteoporosis,
of chemical
degradation
with
peracetic
acid
is whether
the changes
observed
ceasing
to regulate
the composition
to wet
and
before
tissue
the
tissue
final
the
result
became
resorption
on individual
followed
by
collagen
potassium
in osteoporosis
of the fluids
always
in contact
516
K.
LITTLE,
M.
KELLY
AND
A.
COURTS
Section
of unfixed
and decalcified
osteoporotic
bone.
The matrix
has lost its fibrillar form
and appears
as a structureless
mass.
Canaliculi and other fine details can no longer be observed.
(x 4,000.)
FIG.
28
bone from rabbit
with experimentally
produced
osteoporosis.
There
is a
the normal
fibrillar
matrix
and the osteoporotic
matrix
which
has become
as a result of the acid treatment.
(x 4,350.)
Figure
28-Section
of a similar
border
between
the
osteoporotic
regions.
An osteocyte
lacuna can be seen, with canaliculi
and fibrils in its associated
matrix.
The adjoining
matrix
has lost its detailed
structure.
(x 4,000.)
FIG.
Figure
sharply
27-Section
defined
structureless
normal
and
27
of decalcified
border
between
THE
JOURNAL
OF
BONE
AND
JOINT
SURGERY
STUDIES
with
tions.
the
bone
from
be
this
the
work
degrading
of
Sometimes
osteoporosis
1961).
true,
because
At
affecting
the
intervertebral
fractured
disuse
atrophy,
Collagen
as
A
seems
and
collagen
healthy;
also
bone
who
diabetes
in
osteohave
been
listed
to include
a generalised
adaptation,
the
post-
F
mellitus,
adaptation
After
syn-
an early
porosis
in the elderly
more
readily
affected
NO.
3,
AUGUST
stage
of Buhr
and
29
irradiation
of
has
lost
tissue
itself.
Thus,
in disuse
of bones
are affected.
Again,
Cooke
(1959)
skin.
dry
intact, but the ground
together
holdingbndles
FIG.
30
with peracetic
acid, followed
by potassium
in the degradation
of individual
collagen
the observations
than others.
1962
50 megarad
collagen remains
condition
of the bone
only a limited
number
which has been treated
shows
44 B,
so,
rheumatoid
arthritis.
In themselves
to be unrelated.
Often the condition
is not even
post-traumatic
VOL.
be
vitamin
functioning
post-traumatic
be
of osteoporosis
cells
are
starvation,
drome
and
these appear
to
converse
of osteoporosis
menopausal,
that
to
the
(1958),
Urist
osteo-
collagen
of
the
discs
normal
by
degradation
excess
type
produce
causes
have
considered
times
bones
described
517
bones
definite
appears
by
osteoporotic
that
various
this
vertebrae
BONE
can
some
generalised
in the common
in the
OSTEOPOROTIC
Cohen
a probability
a
other
AND
is required.
been
caused
areas
the
with
agent
has
(Fell
with
NORMAL
condithat in
changes
of
is therefore
manifestation
disease.
certain
not
Osteoporosis
blasts
IN
1962,
matrix
associated
There
active
large
(Sissons
the
group-but
collagen
an
one
MATRIX
quite
cells
in
porosis.
in
people
living
observed
from
of
elderly
of
in the present
been
BONE
matrix,
or only under
specific
it has already
been mentioned
devoid
1962);
ON
The
substance
its strength.
atrophy
with
and
osteo
hydroxide.
This photograph
fibrils.
(x 15,000.)
suggest
that
some
bones
are
518
K.
The
variations
in origin
of osteoporosis
start
intermediate
factor
observations
affected
in
it to go
stages
solution
other
the
very
quickly.
of
osteoporosis
types
that
at
the
suggest
final
with
necessarily
There
It
is a need,
produce
has
been
several
that
similar
of the
been
directed
at preventing
that
in those
types
imbalance
look
for
other
have
the
bone
the
to trigger
possible
result,
a stage
while
now
shown
means
and
not
the
a cause,
which
them.
process,
because
of oestrogens
oestrogens
has
in
probably,
condition.
to be the
damage.
osteocytes
understanding
rats
is
in fact,
It is thus
appeared
damage
it does
have
of osteoporosis
the
of
of osteocyte
our
That
balance
administration
of the
early
subsequent
destroying
in
treatment
could
is found,
causes
effect
imbalance
cause
processes
the
one
that
immediate
a process
the
completely
stage
that
a hormone
more
intermediary
If such
disturbance,
This
where
off the
condition.
progressed
final
intermediate
vitamin
A
and caused
in the
causing
all the various
necessarily
be,
by Fell
excess
matrix
damaged
enzymes,
an
has
matrix.
of osteoporosis
served
produce
(1962)
the
it may
experiments
osteocytes
are
breakdown
whatever
that
bone
an
work
collagen
solution
in the tissue fluids.
experiments
on metabolic
by which
was
hormone
Budy
the
active
without
action
involve
effect.
means
locally,
hormone
causes
resorption
to discover
osteocytes
that
a therapeutic
to inhibit
to
thought
apparent
has
therefore,
damage
associated
by the
be
this
a systematic
demonstrated
degraded
causes
could
osteoporosis
their
is suggested
possible
clinical
; this
that the local factor,
on the collagen.
degradation
of the matrix,
which
would
then go into
go into solution
is a point
which
many
elaborate
verified.
could
various
matrix
In disuse
osteocytes
She
enzymes
liberate
the
in the
It is not
degradation
It is thus
that
seen
bone.
is evidence
than directly
osteoporosis.
cells.
These
COURTS
effects
the
a time.
Here
rather
for
the
within
(1957)
A.
of severity
operated
of generalised
enzymes
from
AND
produces
one
mechanism
into
of
which
sometimes
on a cause
proteolytic
KELLY
in degree
of trabeculae.
the osteocytes,
A possible
M.
which
by Frank
patches,
along
the surfaces
operates
through
(1961)
released
and
a process
local
confirms
LITTLE,
possible
cause,
this
We must
now
or
otherwise
of osteoporosis
will
further.
SUMMARY
1.
The
2.
are
In the matrix
in solubility,
appearance
appears
as
of larger
3.
In
fine
of decalcified
fibrils
diameter
bones
bone
matrix
In
from
ancient
fibrils.
5. When
Under
predominates.
elderly
6.
bones
In
subjects
Evidence
osteoporosis
and
decalcified,
conditions
collagen
modified
electron
adult
the
bone
microscope
is described.
distinguished.
In infant
form
which
Differences
bone
the
appears
observed
form
which
as tubular
fibrils
predominates.
the
chemical
eucollagen
sometimes
hydrolyses
fatty
found
on the x-ray diffraction
patterns
4.
in the
two types
of collagen
fibril have been
x-ray
diffraction
pattern
and appearance.
fossils
the matrix
in which
in osteoporotic
x-ray diffraction
has
is some
been
only
the
stable
a small
produced
factor
tubular
in osteoporotic
bone
disperses
pattern.
local
reaction
employed
acid esters,
and lines
of the insoluble
residue
to
bones
fraction
in
suggest
affecting
form
the
of collagen
loses
that
from
acid.
The
the
immediate
osteocytes,
are
but
the
fine
and
normal
not
fibrillar
bone
residue
cause
than
into
acid
survives,
insoluble
rather
collagen
free fatty
extraction.
its architecture
is dissolved
citric
to convert
due to the
after citrate
of
a general
most
then
many
form.
of the
gives
forms
chemical
a
of
effect.
The
and
authors
wish to thank Professor
J. Trueta
not only for the facilities
he gave them, but also for his advice
encouragement.
Their
thanks
also go to Professor
Trueta
and all the other surgeons
at the Nuffield
Orthopaedic
Centre
who gave them operation
specimens;
to Dr W. A. Aherne
and Dr A. J. Buhr for postmortem
material;
and to Dr C. Cooke and Dr A. J. Sutcliffe for ancient
bones and fossils.
Mr J. A. Moss kindly
let them have some of his collagen
samples
for electron
microscopy.
Mrs E. M. Holdoway,
Miss J. Bowerman
and Miss M. Litchfield
provided
invaluable
technical
assistance.
THE
JOURNAL
OF
BONE
AND
JOINT
SURGERY
STUDIES
ON
BONE
MATRIX
IN
NORMAL
AND
OSTEOPOROTIC
519
BONE
REFERENCES
W. T. (1943):
Textile
Fibres
under
the X-rays.
Imperial
Chemical
Industries
Ltd.
J. H., Moss,
J. A., and RAISTRICK,
A. S. (1960):
The Action
of 1.-Rays on Collagen.
Journal,
76, No. 2, 21.
BUDY,
A. (1962): C.I.O.M.S.
Symposium
on Radio Isotopes
andBone.
Oxford : Blackwell
Scientific
Ltd.
BUHR,
A. J., and COOKE,
A. M. (1959):
Fracture
Patterns.
Lancet,
1, 531.
COHEN,
J. (1962): C.1.O.M.S.
Symposium
on Radio Isotopes
andBone.
Oxford : Blackwell
Scientific
ASTRURY,
Bowrs,
Biochemical
Publications
Publications
Ltd.
A. (1959):
Solubilized
Collagen.
Nature,
183, 440.
A. (1960):
Structural
Changes
in Collagen.
Biochemical
Journal,
74, 238.
FELL,
H. B. (1961):
Experiments
on the Action
of Vitamin
A on the Ground
Substance
ofCartilage
and Bone
in Culture.
Journal
of Bone
and Joint
Surgery,
43-B,
I 80.
FRANK,
R. M. (1957):
Thesis.
Strasbourg:
Contributions
a l’#{233}tudeau microscope
#{233}lectronique des tissues
calcifl#{233}snormaux
et pathologiques.
GEISER,
M., and TRUETA,
J. (1958):
Muscle Action,
Bone Rarefaction
and Bone Formation.
Journal
of Bone
and Joint
Surgery,
40-B,
282.
KELLY,
M., LITTLE,
K., and COURTS,
A. (1959):
Bone Matrix
and Osteoporosis.
Lancet,
ii, 1,125.
KENNEDY,
J. J. (1955): Tubular
Structure
of Collagen
Fibrils.
Science,
121, 673.
LITTLE,
K. (1958):
The Use of Stereoscopic
Techniques
in Electron
Microscopy.
Journal
of the Royal
Microscopical
Society,
78, 53.
LITTLE,
K. (1959):
Investigation
of Nylon
“Texture”
by X-ray Diffusion.
British
Journal
of Applied
Physics,
10, 225.
LITTLE,
K., HOLDOWAY,
E. M., and BUHR,
A. J. (1962):
The mineral
phase
in osteoporotic
bone.
(In preparation.)
LITTLE,
K., and KRAMER,
H. (1952): Nature of Reticulin. Nature,
170, 499.
LITTLE,
K., and WINDRUM,
G. M. (1954): A Lipid Component
of Reticulin.
Nature,
174, 789.
SlssoNs,
H. A. (1962):
C.I.O.M.S.
Symposium
on Radio isotopes
and Bone.
Oxford:
Blackwell
Scientific
Publications
Ltd.
URIST
M. R. (1958): The Problem
of Osteoporosis.
Clinical
Research,
6, 377.
COURTS,
COURTS,
44 B,
VOL.
E
NO.
3,
AUGUST
1962