mineralization in bone, dentin and turkey leg tendon. Evidence for the close ...... collagen. Various macrornolecules such as phosphoproteins. (Veis, 1 985b), y ...
A C O R R E L A T I O N BETWEEN THE D I S T R I B U T I O N OF B I O L O G I C A L AND AMINO A C I D SEQUENCE OF T Y P E I C O L L A G E N
APATITE
By MURRAY E.
MAITLAND
B.S.R.(P.T. and O.T.), U n i v e r s i t y of B r i t i s h Columbia,
A THESIS
1984
SUBMITTED IN P A R T I A L F U L F I L L M E N T REQUIREMENTS FOR THE DEGREE OF MASTER'S OF S C I E N C E
OF
THE
in THE
We
accept
this
F A C U L T Y OF GRADUATE S T U D I E S (Department of Anatomy)
thesis
THE
as c o n f o r m i n g
to the require
U N I V E R S I T Y OF B R I T I S H COLUMBIA M a r c h 1989 ®Murray E. M a i t l a n d
standard
In
presenting this
degree
at the
thesis
in
University of
partial
fulfilment
of
of
department
this or
thesis for by
his
or
requirements
British Columbia, I agree that the
freely available for reference and study. I further copying
the
representatives.
an advanced
Library shall make it
agree that permission for extensive
scholarly purposes may be granted her
for
It
is
by the
understood
head of
that
copying
my or
publication of this thesis for financial gain shall not be allowed without my written permission.
(Signature)
_
Murray E. Maitland
Department
of
A
n
a
t
o
m
y
The University of British Columbia Vancouver, Canada
Date
DE-6 (2/88)
A p r i l 25, 1989
i i
ABSTRACT
The
present
distribution fibrils of
of
of
was
dark
bright
field
both
electron
data
computer
microscopic
in
both
the
computer
images
was
density. on
density of
The
and
C-
of
on
the for
gap
known
but
each
mineral
proportion
of
of
hydrophobic
amino
acids.
hydrophobic
e f f e c t s and
The the
are
discussed.
In
addition,
the
deposition
of
apatite
in
with
chick These
apatite
early
stages
corresponds
low
in
of
the
of
collagen
hydrophobic the
overlap
areas
of
corresponded regions
density
with
mineral
accommodation zone
high to a
of
interactions
overlap
to
deposition
than
had
which
of
theoretical
high
zones
process
the
that
mineral
of
possible
the
from
sequence
Conversely,
were
was
modelling.
at
hydrophobic
acids
density.
mineral
of
areas
these
amino
and
and
which
acid
stages
is less
hydrophobic
low
amino
from
collagen
orientation within
comparisons
early
zone
pattern
molecular
Based
zones
I
acids
illustrated
overlap
asymmetric
i s excluded
average of
gap
to
determined
apatite
techniques
i n an IM-
by
amino
the
localization
microscopy
sequence
fibril.
high
The
I collagen
collagen
sites
by
type
tendons.
of
polarized
zone
field
leg
within
classes
mineralization
that
turkey
examine
specific
occurs
it
apatite
to
with
electron
the
undertaken
determined
selected-area correlated
was
biological
calcifying
apatite
type
study
between
deposition of is
collagen
to
discussed.
i i i
TABLE
OF
CONTENTS
Page
Numb n
Abstract Abbreviations List
of Figures
Acknoiuledgements Historical
Review
Introduction Materials
and
v y i 1 28
Methods
32
Results
37
Discussion
gg
Conclusions
58
References
7Q
iv
LIST
Figure
OF
FIGURES
Title
Page
Number
1.
Hodge
and P e t r u s k a
2. E l e c t r o n turkey
micrographs
5. S t a g e s
39
of g l y c e r o l
of c a l c i f i c a t i o n
Analysis
Electron and
9.
of collagen
alcoholic stained
hydrophobic
10.
the
i n collagen.
50
orientation
tendon.
diffraction
A comparison
near
46
7.
mineralized
42
treated
front.
L o c a l i z a t i o n of apatite
8.
fibrils.
45
6.
in
of collagen
tendon.
mineralization
16
non-mineralized
staining pattern
Lou m a g n i f i c a t i o n turkey
of
packing model.
l e g tendon.
3. P e r i o d i c
4.
(1963)
54
i n unstained mineralized
of the d i s t r i b u t i o n amino
acids
D i s t r i b u t i o n of p r o l i n e
mineralized tendon.
54
of
and m i n e r a l
apatite.
and h y d r o x y p r o l i n e .
59
59
ABBREVIATIONS Amino
Acids
A
Alanine
K
Lysine
R
Arginine
C
Cysteine
L
Leucine
S
Serine
D
Aspartic
Acid
1*1
Methionine
T
Threonine
E
Glutamic
Acid
N
Asparagine
U
Hydroxylysine
Phenylalanine
0
Hydroxyproline
V
Valine
F G
Glycine
P
Proline
Ui
Tryptophan
H
Histidine
Q
Glutamine
Y
Tyrosin
I
Isoleucine
Other
Abbreviations
a
" a " bands
of collagen
staining
pattern
b
"b" bands
of collagen
staining
pattern
" c " bands
of collagen
staining
pattern
c
-
C-
Carboxy
d
" d " band
D
Axial
e
"e" bands
h
Hour
min
Minute Amino
terminus
of peptide
of collagen
period
of type
of collagen
terminus
nanometer
PTA
Phosphotungstic
SADF
Selected-area
UA
Uranyl
jqm
Micrometer
Acetate
staining I
pattern
collagen staining
of peptide
nm
chain
pattern
chain
acid
dark
field
electron
microscopy
vi ACKNOWLEDGEMENTS A t t h i s t i m e I w o u l d l i k e t o t h a n k t w o i n d i v i d u a l s who w e r e i n s t r u m e n t a l i n my c o m p l e t i o n o f t h i s t h e s i s . D r . A r s e n a u l t has c r e a t e d a wonderful environment w i t h i n h i s l a b through h i s p a t i e n t guidance and s u p e r v i s i o n . A l s o , h i s c r e a t i v e i d e a s and p r o d u c t i v i t y a r e c h a r a c t e r i s t i c s which i n s p i r e a n d m o t i v a t e . L i s a , my w i f e , w a s l a r g e l y r e s p o n s i b l e f o r t h e s u c c e s s o f t h i s e n d e a v o u r b e c a u s e s h e c o n s i d e r e d my g o a l s i m p o r t a n t e n o u g h t o e n d u r e my many y e a r s a s a s t u d e n t . In a d d i t i o n , I would l i k e t o c o l l e c t i v e l y thank the' g r a d u a t e s t u d e n t s , p r o f e s s o r s a n d r e s e a r c h e r s who s u f f e r e d my many q u e s t i o n s a n d a s s i s t e d me i n l e a r n i n g t h r o u g h o u t t h i s program. F i n a n c i a l l y t h i s w o r k was s u p p o r t e d by a g r a n t t o D r . A.L. A r s e n a u l t f r o m t h e M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a a n d a s c h o l a r s h i p t o M. E . M a i t l a n d f r o m t h e P h y s i o t h e r a p y Foundation o f Canada.
1
HISTORICAL
REVIEW
Introduction As loose most
a major
component
and dense
connective
prevalent
importance in
works
of this
about
their
collagen
These
collagen
structure,
works
research
have
on c o l l a g e n that
directly
related
entire
world
turkey
tendon
development,
this
review
of m i n e r a l i z a t i o n
some
for this
i n order
to avoid
formed
previous
type
areas
to
The v o l u m e o f tissues which are
explore
i s a model
the since the
o f bone
differences In t h i s
specifically
unneccessary
collagen
i n order
However,
be d i s c u s s e d .
refer
I
several
i n various
and
current
protein
area.
research.
structure
our
microscopy
than
but i t s The
apatite
within
i n this
study
will
will
understood.
morphology,
of the s i m i l a r i t i e s
t h e two t i s s u e s
protein,
have
rather
i s the
has r e s u l t e d
be s e l e c t i v e o f p a p e r s
to the thesis
used
processes
c o l l e c t e d from
of ideas
of
I t s recognized
structure,
and e l e c t r o n
types
I collagen
i s t o examine
and m i n e r a l i z a t i o n
use of " c o l l a g e n "
collagen
been
including
biochemistry,
requires
the
poorly
review
and other
this
mineralization
the development
between
about
i n t e r - r e l a t i o n s h i p s which
fibrils.
works
remains
molecular
of b i o l o g i c a l
assess
of b i o l o g i c a l
historical
concepts
of
t i s s u e s , type
of information
i n mineralization
purpose
tendon
i n vertebrates.
i n a variety
an a b u n d a n c e
role
and
protein
of bone,
thesis
to type
I
r e p e t i t i o n . The
term
2 "biological
a p a t i t e " i s used
differences
between
model
for apatite
biological
recently 1 984;
hierarchy
molecule;
collagen
collagen
many
fibers
have
wishes
been
best
and t h e a c t u a l further
published
and Hulmes,
1984; Bonnucci,
responsible different
consist
from
tissues, collagen
from
chains
the molecular form
molecules
microfibrils
with
form
o f many
other
molecules
These
to the
combine
t o form
a collagen
f o r microns diameters
o f t h e e y e t o 500nm
a l . , 1978).
extracellular
In bone, matrix
network,
a
fibril;
Differences
and
result
of the
extracellular
prior
properties collagen
to
support
from
i n mature
collagen
of the
fibrils
or centimeters
vary
of osteoid
and
architectures are, i n part,
t i s s u e s . For example,
tissue. Fibril
has a
the collagen
fibrils.
f o r the biomechanical
extend
tissue
post-translational modifications
i n t h e body.
a n d may
defined
and d i f f e r e n c e s
r e s u l t i n g i n a v a r i e t y of architectures
tissues
humour
order
peptide
tissue-specific
matrix,
et
reviews
a l l connective
several
interactions
the
good
I f the reader
has s i m i l a r i t i e s
Three
microfibril;
long
structure.
of structural
microscopic.
the
tendon,
1 9 8 4 ; Chapman
tissue. Within
from
c r y s t a l s i n turkey
the
K'uhn, 1 9 8 7 ) . Collagen
to
t o emphasize the
considered
several
(Miller,
thesis
hydroxyapatite,
crystal
information
i n this
1Onm
depending
on
tendon
(Parry
within the
are interwoven,
to mineralization.
very
i n the vitreous
rat tail
fibrils
are
in a
Following
poorly
3
remodelling within
the
of
bone
are
shear
lamella.
fibrils
close
to
within
90
bone,
parallel
"twisted
the
fibrils
which
fibrils
one
present, fibrils
the
within
sinusoidal fibrils
may
In
course,
forces. rapid
The
minimum Turkey
of of
leg
the
lamella
within
nested to
to
arcs.
of
muscle.
organized The
forces
and
which
this
into
a
is model
the
patterns of
They
a
follow occur
collagen
and
as
tensile
tendons
by
these
deformation
permit
contractile loads
(Silver, a
1 988).
parallel, high
of
by
are
(Giraud-Guille,
generated
mineralize
In
arrangement
sustain
transmit
angle
continuously
general
contain
of
fibrils
d i r e c t i o n of
rotates
two
an
same s p e c i m e n ,
fibrils.
composition
Tendons
loss
tendons,
and
can
direction
of
direction
which
are
organization
complex.
tendons
into
by
h e l i c o i d a l bundles
bone,
structure
the
the
next
of
are
these
to
the
another
change
fibrils
energy
and
lamella
local
transmission
elements
another
i s very
abruptly
densely-packed
one
three-dimensional
bone
contrast
fibrils
Although
local
organized
the
architecture
lamella
angle.
are
fibrils
In
lamella
occur
of
Collagen
d i f f e r e n t forms.
fibrils
one
collagen
r e f l e c t i o n of
alternative
each
series
from
An
plywood"
superimposed
constant
from
of
each
to
may
within
is a
two
model,
degrees.
architecture
distractive forces.
Within
changes
the
a
matrix
and
plywood"
predominantly the
laminated
distributed in
"orthogonal distinct
the
mineralized
compressive, in
bone,
part
with
a
1987). of
normal
the
4
growth
after
9 weeks
of
age,
have
microscopic
i n v e s t i g a t i o n s of
because
linear,
the
analysis and
along
Engstrbm,
individual 1965;
Collagen, also
Evidence
for
biological from
of
i n bone,
the
close
of
defects
the
mechanisms The
primary
give
composition integrate
of new
gap
zones
these
of
In
leg
of
and
of
function
most
a
stems
are
the due
part,
about
skeletal to
1987).
function
work to
i s the
the
from
amino an
of
the
concepts
Recent
acid
information within
stages
1989).
As
an
need
to
of
(Arsenault,
early
localization
apparent
apatite crystals
(Arsenault,
in
relationship.
current
fibrils
and
heterogeneous
(Hollister,
i s known
this
collagen
instances,
i n the
this
i n the
Myers
tendon.
comes,
imperfecta
respect
collagen
zones
turkey
imperfecta,
interaction.
gradient
a l . , 1960;
of
bone
to
findings with
distribution
developmental within
with
collagen
collagen/mineral
et
p h y s i o l o g i c a l process
collagen
rise
permits
is
and
little
interest
apatite crystals
fibrils
role,
I collagen
of
t i s s u e s very which
(Nylen
of
r e l a t i o n s h i p between
importance
mineralized
and
dentin
type
electron
i t s biomechanical
osteogenesis of
in
1988).
the
inherited diseases.
molecular
periodic
in
osteogenesis
m a n i f e s t a t i o n s " of
Despite
fibrils
a p a t i t e i n normal
studies
group
arrangement
Arsenault,
intimately involved
used
collagen mineralization
i n a d d i t i o n to
mineralization
of
parallel
been
suggests the
1988),
a
overlap and
a
of m i n e r a l i z a t i o n extension
of
5
these the
works
the present
study
periodic distribution
of apatite.
structure
and t h e p r o p e r t i e s o f b i o l o g i c a l
The
given
Collagen The
chains and
helical Crick,
interact
apatite
of t h i s
c o n s i s t s of three
t o form
1955; Rich
chains
and a r e c o i l e d
1955).
Piez
a triple
and C r i c k ,
polypeptide
coil
proportions
molecular
thesis.
e t a l . (1963)
of the three unit
peptide
helix
have
each
determined chains
individual
( R i c h and the r e l a t i v e
and d e s c r i b e d t h e
molecular
peptides
and one
2(l) peptide.
collag'en
molecule
was f i r s t
described
by B o e d t k e r
(1956) as a r i g i d
r o d 300nm
i n length
and h a v i n g
1.25nm. L a t e r , when
resolved, one
i t was
a t each
terminal both These
found
as c o n s i s t i n g
et al.,1955).
their
other
t h e amino there
t h e N- a n d Cconformations
prediction
based
and shape
sequence
1(1)
of the and
a
Doty
diameter
was
being
a r e two n o n - h e l i c a l p o r t i o n s ,
end o f t h e m o l e c u l e .
portions called
o f two i d e n t i c a l
The s i z e
acid
peptide
(Ramachandran
1 9 5 5 ; Cowan
each
about
helical
collagen
of
crystals
Molecule
which
three
of collagen
t o the development
collagen molecule
Kartha,
These
rise
analyze
I t i s therefore
to review
have
concepts
to further
necessary
which
some
attempts
These
are the
a n d C-
t e l o p e p t i d e s . The c o n f o r m a t i o n s
t e l o p e p t i d e s a r e n o t known a t are modelled
on e n e r g y
using
and s t e r i c
secondary
of
present. structure
calculations.
Several
6
stereochemical this
method
models
(Jones
may
and
of
may
not
the
be
semiflexible molecules on
their
side
would
the
within
the
interactions. dependent pitch
will
Although is
on
the
The
pitch
be
between
or
the
to
et
of 29 al.
a
single
rat
tail
tendon
45
residues
and
(1983)
value
of
the
the
the
side
chains
be of
the
edge.
collagen
molecule
intramolecular agreed
been
1976).
pitch
acid
intramolecular
been
has
(Miller,
calculated
acid
reaction
yet
edges
chains
would
and
collagen
portion
amino
side
the
the
not
rigid
a
alteration
inter-
has
as
of
amino
any
pitch
a
determine
edges
of
as
reaction
acid
would
the
and
the
via
position
the
of
the
determining
other
determine
pitch
of
from
cylindrical
amino
composition
determination
interactions,
Fraser
helical
of
while
would
regions
helical
described
cylinder
composition
change
critical
the
helix
The
triple
Two
the
groups
of
these
molecule
be
each
on
interactions
triple
The
also
with
depending
exterior
collagen
(K'uhn, 1 9 8 2 ) .
Functional
intermolecular
can
interact
surfaces
the
for
1987).
correct.
molecule
cylinder
chains.
towards
of
entirely
collagen
possible
Miller,
Conceptualization rod
be
to
upon.
reported The
be
study 30
to of
residues
8.68nm. Individual
flexibility fluctuations without
collagen
(Veis, are
molecules
1985a).
This
accommodated
appreciable
changes
in
can
by
show
occur
changes
free
substantial because in
eneregy
the
lateral segmental
triple
(Okuyama
helix
et a l . ,
7
1981).
Flexibility
motions
and
regions
located
they
are
2)
hinge
the
stability
about
the
N-C
restricted.
because
Thus,
of
flexible the
simplistic.
backbone
and
suggest
that
involve
a
domains.
myriad
of
et
collagen
of
molecule
as
molecular flexibility
of
the
molecules
these
long
axis
and
38Kb
is
by
is a
two
of
et
the in
peptide
1987)
molecules
in
fluid-like cause
transverse
axis
more
and
fibrils
in mineralization I
issues
of
the
local
explore
relevant
fully
of
in
I collagen length
of
are
which
the
properties would
protein translation
Genes type
be
NMR
a l . ,
would
also
to
T y p e _I C o l l a g e n
about
there
adjacent
changes
The
are
rotation
c y l i n d e r may
changes
post-translational modifications.
for
a
Sarkar
and
genes
in
C-C=0 b o n d
flanked
observed
like
The
which
element
molecule
motions
conformational
order
discuss
because
prevents the
length
now
They
to
In
250nm
i n t e r a c t i o n s between
i n both
helix.
collagen
hinge
residues
major
structure
a l . , 1983;
Accommodation
distortions triple
the
The
chain
(Sarkar
is a
flexural
915-939
hydroxyproline
r o t a t i o n about
about
slow
suggested
81-102 and
ring
the
collagen
side
1)
regions.
somewhat
experiments
the
forms (1984)
Proline
the
within
region
Viewing
residues
helix.
and
two
Veis
p r o l i n e and
bond
semiflexible relatively
of
in
regions.
triple
helix
occur
between
devoid
stabilize
may
large 10%
and
is a
complex. coding
a sequence many the
o f a b o u t 50 e x o n s
exons have ancestral
(K'uhn, 1 9 8 4 ) .
an i d e n t i c a l
gene
arose
o f 54bp
unit
sixfold
r e p e t i t i o n of a oligonucleotide
pattern
sizes
recombinational
may
events
shows
molecule
b a s e d on " b i o c h e m i c a l of collagens
to
acid
amino
acids
peptide, amino
acids
several
Seyer
bovine acids
acid
chick
I
of the
collagen
species
which
(1985) used
type
a n d human
I
collagen
collagen.
They
(R a n d K; s e e letter
amino
were
codes),
retained.
acids,
and 66% o f t h e h y d r o p h o b i c Overall
amino
microscopic
and Kang
single
i n the a l ( l ) peptide
unsubstituted.
to
91/5 o f
(D a n d E ) a n d 7 9 % o f h y d r o p h o b i c
96% of t h e b a s i c
acids,
this
Nordwig and
of the type
t o compare
amino
f o r amino
a c i d i c amino
of
i n protein
species.
and e l e c t r o n
level.
data
100% of the b a s i c
"Abbreviations" the
structures
a combined sequence from
found
a
f o r the
f o r the invariance
i s o l a t e d from
i n evolutionary amino
between
evidence
and q u a t e r n a r y
available
coding
from
degree of conservation'in
tertiary
differ
arisen
et a l . , 1985).
a high
(1969) presented
studies
have
due t o an i n t o l e r a n c e
sequence and s t r u c t u r e
Hayduk
may
r e f l e c t e d by a c h a n g e
(De C r o m b r u g g h e
Collagen acid
i n turn
that
single
g l y c i n e - p r o l i n e - p r o l i n e . The f i x a t i o n
o f exon
function
which
that
suggests
by a m p l i f i c a t i o n o f a
genetic
tripeptide
o f 54bp
length
The f i n d i n g
homology
between
In thea2 ( l )
87% of the a c i d i c amino
acids
were
the sequence of
9
the a l ( l ) and
calf
chains was
of
found
Consistency prerequisite inorganic for
this
from
a
comes n o t
Amino
Following be and at
subdivided sequence the
which
reticulum
of
the
sites
regions
the
the N-
may
between
be
changes
matrix.
or
alterations. the
a
2
(Hollister,
chain
but
These be
also
such
genetic
and
and
Evidence
formation
i n the
a
organic
i n t e r s p e c i e s homology
Small
rat
a l . , 1982).
code
p a t h o l o g i c a l bone
as
code
changes as
small
as
1987).
the
The
These
first
p r o t e i n to
adjacent
carboxy
because
propeptides. C-
are
from
from
differing
is a the
the
across
peptides
described
region
move
It i s cleaved
with
and
procollagen
six functional regions
It i s considered
along
by
from
of
terminus.
molecule, of
extracellular
transcription,
1981 ) . The
assembly
of
f o l l o w i n g movement
propeptide.
genetic
interaction
the
et
from
Sequence
(rER).
immediately (Kriel,
collagen
the a 1
into
enables
sequence
(Highberger
characteristics.
amino
combined
normal
mutation
Acid
a
biomechanical
either
point
and 93.4%
the
only
studies
i n major
single
be
imperfecta.
involve
The
of
components
osteogenesis
may
to
f o r the
genetic
results
chick
rER
functional region i n the
propeptide,
crossbridges Following
propeptide
starting
signal
rough
size
peptide
endoplasmic
propeptide
the
important
in
can
at
are
the
specific
the
membrane i s the
amino
molecular opposite
end
formed
at
specific
cleavage
of
these
proteinases
in
the
10 extracellular
matrix,
crossbridges.
Adjacent
telopeptide, length.
in
length The
a
(Miller, amino
The
acid
length
The
stages
following
amino
triplet
acid
position helical
D period,
responsible
structure
repeat
acid
except
suggested
stereochemically position
was
more
residues
i s characterized
with
repeat where
234 a m i n o
acids i n
s t a i n i n g and a l s o
I t i s considered
organization.
i n every
i s responsible
sequence
analysed
The
third
f o r the and
triple Kartha,
i s often
or tryptophan.
Jones
t h e X and Y p o s i t i o n s
involved
Other
region
t o be t h e
G i s g l y c i n e , a n d X o r Y c a n be a n y
i n the t r i p l e
interactions.
i n early
t i s s u e , due t o t h e
glycine
of t h i s
cysteine that
by
are f u n c t i o n a l l y
for fibrillar
chain
t h e X p o s i t i o n was
were
in
acid
i s 25
of the molecule- (Ramachandran
The t r i p l e t G-X-Y
16 r e s i d u e s
i s v i s u a l i z e d i n the electron
of a p a t i t e .
of the peptide
abbreviated
while
which
heavy metal
distribution unit
(1985)
patterns
repeating
structural
amino
telopeptide
of m i n e r a l i z a t i o n , i n unstained
periodic
t h e amino
i s 1014 amino
sequence of collagen
(Hulmes e t a l . , 1973),
microscope
1955).
carboxy
segment,
disulphide
1984).
v/ariety of repeating
significant.
portion
t o form
propeptide,
non-helical
helical
i n length.
remains
t o t h e amino
i s a short
The t r i p l e
residues
no c y s t e i n e
helix
such
differ
that
i n intermolecular
involved
more
potential repeating by Hofrnann
et a l .
the Y
interactions
i n intramolecular units
o f amino
e t a l . (1 9 8 0 ) .
They
acid found
11
several D,
2D,
patterns 3D,
unlikely
D/3,
to
functional been and
occur
and
place
may
for
of
to
polypeptides reticulum,
modify
molecular
procollagen
the
the
large
number
and
to
to
and
the
units
are
have
some
has
long
sequence
biomechanical
yet
peptide
there the
has
amino
exist
site
of
i n d i v i d u a l peptides
with
not acid
three
in
while the
1982).
but
of
are
organization
also
of
endoplasmic
Th i s
conformation
hydroxylation.
is a
(Chorpra
of
The
conformational
i s necessary
only
unhydroxylated
conformations
Hydroxyproline not
individual
the
1978).
proline
which
the
rough
g-turn
chains
stabilization
molecule
events
modifications
fibrillar
segments
hydroxylation
the
Ananthanarayanan,
several
These
for
Ananthanarayanan,
proline
of
the
protein.
of
of
collagen
acid
collagen
assembly,
of
i n the
presumed
importance
assembly,
result
role
are
r e l a t i o n s h i p between
associated
specific
intertwining
statistically
amino
of
repeat
Modifications
molecules
(Brahmachari
the
and
The
biomineralization.
molecular are
a
the
chains.
4D/11
t r a n s l a t i o n of
influence
Prior
be
chance
paramount
Post-Translational
may
and
functions
which
essential and
peptide
mineralization.
Following take
D/13
by
of
analysis
sequence
two
s i g n i f i c a n c e . The
considered
an
the
D/2,
physiologic
been
in
i t s assembly
p r i o r to
the
and
plays the
change
an
important
triple-helical into
microfibrils
1 2
(Nemethy always
Scheraga,
i s the
portion that
and
1986).
Y position
(Bornstein
hydrogen
and
bonds
(Gly-X-Y)
Traub,
between
and
et
Hyd r o x y p r o l i n e
the
1975).
structural
(Prockop
bonds the
the
The
between
degree tissues
of and
and
glycosylation
is
immediately
endoplasmic
to
the
polypeptide at
t o be
glycosylating
prior
of
D-galactose
0-glycosidic appears
between (Bansal
essential
a t body
for
temperature
et a l . (1973)
of
the
and
has hydrogen
further
stabilize
ages.
The
role
triple
linkage a high enzymes
degree due
to
and of
of
1980).
the
of
Hydroxy l y s i n e
formation at
to
residues
v i a an
Cunningham,
1966).
few
specific
continue
the
covalently
for
the
1973).
modifications
specificity
is
crosslink
et a l . ,
are
helix
are
lysine
disaccharides
hydroxylysine (Butler
of
helix
(Kivirikko
D-glucose
triple
importance
(Kivirikko, to
to
residues
hydroxylation
mono- a n d and
prior
lysine
post-translational
attachment
molecule.
suggested
water-bridged
i s i n the
reticulum
Intracellular the
collagen
number
formation
with
been
t o be
is
helical
occur
h y d r o x y p r o l i n e which
to collagen
rough
triple
adjacent helix
i s known
specific
hydroxylysine
formed
an
peptide translation
a variable
hydroxylated. variable
on
found
helix.
Following formation
with
of
of
the
I t has
Ramachandran
existence
associated
triple
1976).
1979).
group
integrity
et a l . ,
suggested
a C=0
of
triple-helices
hydroxyproline al.,
H y d r o x y p r o l i n e when
attached
There
the sites
to
which
1 3
the
carbohydrates
found
no
studies
glycosylation
of
attach
(Miller,
directed
at
the
collagen
i n bone
1976).
To
function
date
of
I
have
the
f o r m a t i o n or
mineral
deposition.
Self
assembly Following
extracellular and
secretion
environment,
asymmetrical
molecules
are
the
collagen
the
molecular
of
where
molecules
organized fibrils
into
and
believed
polymerize
Dirk,
1958).
Furthermore,
form
forms
are
dissolution
side
Thus,
chains
directing
on
stabilization side and
chains positive
1974).
been
adjacent
collagen of
molecules
fibril
due
charges
Alternating
to
assembly
have
the
i n the
charges,
G-X-Y
and
a major
Ionic
and
native subsequent
et
role al.,
and
among in
1973)
clustering
position
triplet
positive
the
collagen,
interactions
(Hulmes
relative
in
(Schmitt et a l . ,
that
structure.
of
of
are
(Gross
s p a c i n g and
conditions
suggested
collagen
occurs
by
specific
i t has
fibrils
in vitro
fibrils
1953).
local
the
long
under
the
studies
l o n g s p a c i n g , segmented
of
function
in vitro
interconvertible
these
a
on
fibrous
reaggregation
1985a),
be
within
to
the
elongated
Collagen molecules
spontaneously
based
into
architecture
initially
1988).
as
(l/eis, The
solubility
(Giraud-Guille,
matrix
behave
fibrils.
d e r i v e d may
geometry
extracellular
they
in solution
environment to
procollagen molecules
(Doyle
of
and
of
negative
et a l ,
n e g a t i v e , may
occur
1 A
linearly
to
provide
three-dimensions molecules have
hydrophobic
less
this
The
of
an
groups
aggregation
environment
which
association
of
the
in
i n the
of
c r o s s l i n k s . Hulmes by
orderly
1975).
than
arrangement
of
ions
periodic
spacing
(Piez
and
groups
would
lead
polar
them
collagen (1983)
prior
to
that
concentric
the
1980).
would
suggested
of
of
(Tanford,
fibril
and
addition,
forces
acids
In
two
In
amino
accretion
Torchia,
crystal.
strong
hydrophobic
stability
and
hydrophobic
surrounds
to
occurs
an
of
(Piez
r e s u l t i n more
ionic
occur
d i s r u p t i o n of
growth
would
c o n t r i b u t i n g to
properties
1978).
stability
Trus,
aqueous The
close
therefore the
lead
formation
collagen
layers
to
of
fibril collagen
molecules. Procollagen
processing
interactions
with
suggested
as
factors
diameter.
Aminopropeptides
and
may
1983). been
regulate
Procollagen
(Nowack
et
formation
may
on
alternative
the
tissue
and
hypothesis
to of
by
Procollagen
the
the
control
been fibril
et a l . ,
I collagen
have
immunofluorescence
polymerization
remove
or
fibrillogenesis
type
a l . , 1983).
a l . , 1985). fibrils
of
immunoelectron
the
have
(Fleischmajer
fibrils
et
a l . , 1983)
specific
participate in
collagen
involve et
the
diameter
(Fleischmajer
(Fleischmajer act
limiting
et
(Wood, 1960)
aminopropeptides
a l . , 1976)
techniques
then
proteoglycans
fibril
demonstrated
(Fleischmajer
microscopic Therefore, of
aminoprocollagen
N-proteinase
amino of
fibril
would
propeptide.
fibril
size
is
An
1 5
derived
from
in vitro
interactions
which
related
to
present
(Parry
The
the
period
due
to
1952).
Early
determined al.,
values et
al.,
al.,
changes
in D
dependent
The
in fibril
type
pattern
feature
which of
of
the
x-ray
true
collagen but
local
D
charged
period
the
degree
i t may
are also
collagen
aggregates
(Hodge
and
led to
(i960) defined
for
collagen
thesis.
has
Hodge
and
fibril
the
longitudinally
space
(see Figure
1).
(Bear,
(Hall
yield
to
et
greater (Fraser in
artifactual specific
or
of
repeating
the
in theories
In a d d i t i o n ,
" a , b,
banding
units
This
of
of the
electron
quarter-staggered packing
c,
which
P e t r u s k a (1963)
which
nm
remains
tissue
resulted
Schmitt, 1960).
Schmitt
acids
stretch
two-dimensional packing arrangment. approach
D
environment.
vitro
microscopic
i s the
collagen 64
unit
be
microscopic analysis
the
of
prone
electron
model
of
methods
value for this fibrils
collagen
amino
t o be
diffraction on
of
r e p e a t s i n each
The
molecular
diameter
of p r o t e o g l y c a n
microscopic studies
length
the
and
localization
length
on
changes
1982).
staining
However,
because
proteoglycan/collagen
ultrastructural
electron
1983).
of
found
(67.8nm) d e p e n d i n g
debate
in
et
the
the
1942).
have
concentration
striking
cross-striated
studies
d and
e"
will
be
first
Hodge
nomenclature used
suggested
a d j a c e n t m o l e c u l e s were
and
in
this
a model
s e p a r a t e d by
in a
1 6
D=67nm
D=234
OVER| P ZONE
GAP, ZONt
RESIDUES
A
•'.•'.•'.•'.•'.•'A
it
•
^••••••••Vr]
ESsd
•>::.:>>::v^^
t
K-WX-K
AD
.6D 40.2nm
26.8nm
44D=300nrn
Figure type
rods
staggered
adjacent
different packed
densities
overlap
available.
zone
Second,
be a n a l y z e d
sequence for
First,
repeating model.
These
This
t h e model within
units
are depicted as
r e s i d u e s , o r D,
theperiodic
sequence
to
has two i m p o r t a n t
predicts
a n da gap zone
according
acid
model o f
two zones o f
therepeating D unit,
by computer
acid
packing
molecules
model
with
thesis
using
o f c o l l a g e n were
densely space
o f amino
acids
the primary
thesingle
t o t h e Hodge
a
potential
localization
modelling
ofcollagen. Inthis
t h e amino
(1963)
by 234 amino
molecule.
consequences.
can
andP e t r u s k a
I collagen molecules.
linear the
1. T h e Hodge
letter
organized
and Petruska
codes into
(1963)
17
The
Hodge
consequence different contain would the
and P e t r u s k a
that
contain
A closely
fitting
(1963)
four
between
has been
model
the electron microscopic
fibril be
amino
used
i n this
microscopic model
acid
(Meek
Despite
and r i g i d .
the i l l u s i o n
In r e a l i t y
There
the l e v e l
about axis the
of the f i b r i l .
the molecular a n d i n some
fibril appears
of
t h e gap zone
However, this
illustration
the c o i l
about
o f t h e way
to the scale
the width
gap z o n e s
this
molecules
levels
the
of
in coiling
the
coil
microfibrillar
are coiled
within
In a d d i t i o n , i n Figure
Figure
holes 1 was
at the
( o r 0.5mm). So small
based
1,
level
constructed.
of the a c t u a l molecule,
o f t h e gap zone
i s very
will
that the
of the chain,
are l a r g e , porous
of the molecule
individual
coil
the m i c r o f i b r i l s
because
t o be m o r e
or four
et a l . , 1979).
as i f there
x the length the
cases,
(Ruggeri
it
axis,
The
This
the
space
are three
and
to the current
s t r u c t u r e of
s t r u c t u r e of the collagen
to
Hodge
i n electron
three-dimensional i s complex.
The
of
e t a l . , 1979) which
i n v e s t i g a t i o n s the f i b r i l l a r
are linear
because
analysis of collagen
i t s utility
i s oversimplified. I t gives
molecules
zone
s t r u c t u r e and h i s t o c h e m i s t r y .
composition
study.
would
gap
molecules
important
of collagen
of
zone
molecules.
very
important
zones
overlap
understanding permits
has t h e
molecules.The
f o r every
separation model
packed
parallel
one s p a c e
longitudinal
Petruska
model
i t p r e d i c t s two a l t e r n a t i n g
densities.
tightly
(1963)
would
be
5/1000
t h e volume on t h i s
in
of model.
1 8
For
example,
shaped sides of
space 1.5nm
i f we with
this
about
3,000nm^,
see below)
gaps,
two i d e a s
have
aligned
i n three
(Weiner
and Traub,
arrangment order.
Second,
built
the
primary
zones.
into
they
Current
been The
H'ohling
views
of crystal
fibrils
exhibit
support
the concept
may
be
space
predict
(1963)
packing
n o t be i n t h e that
staggered
larger
spaces
and n o t t h e gap
dimensional
packing
theories but neither
packing
crystalline
order.
qualities
of periodicity
have
There
may
of collagen
i n
several
In c r o s s - s e c t i o n i n some
regions
which
three-dimensions.
a r e n o t c o n s i s t e n t . I n some
the cross-section pattern areas.
arrangement
i s n o t known d e s p i t e
portions of the f i b r i l
irregular
individual
demonstrated.
at defining a regular
However,
would
formation
these
of
300nm^ t o
o r g a n i z a t i o n and these a r e
of the three
do n o t e x c l u d e
to explain the
sufficient
suggested
the m i c r o f i b r i l l a r
within the f i b r i l
attempts
theory
would
(1 9 8 0)
three-dimensional
molecules
regions
molecules
location
arrangement
to provide
volume
non-collagenous
t h e gap z o n e s
i n t h e Hodge and P e t r u s k a because
are
First,
This
the total
(minimum
and t h e volume
dimensions
and t h e o t h e r
In order
of mineral
emerged.
1986).
40nm
no o t h e r
space.
the volume
gap as a box
1963) then
be 90nm^ a s s u m i n g
between
distortions
dimension
and P e t r u s k a ,
are occupying
difference
the individual
the longest
(Hodge
one gap w o u l d
molecules
consider
i s distorted
a l s o be s h a r p
over
discontinuities
to
1 9
the
lattice-like
close
packed,
optimal
quasi-hexagonal Miller, 1976)
1979)
may
a
consistent regions
fibrils
i s considered
crystal
since
growth
to
evidence
distance
Katz to
collagens. in
vary They
needs
reasons.
different
in
crosslinks
to
and
Li
short not
be
between found
tissues
In
the
appropriate
(Mechanic
et
the
collagen
apatite
environment have
crystal
There
organization
is
is
some
tissue
intermolecular
and
non-mineralized spaces
non-mineralizing
inhibited in
tissues
to
of
of
volume.
intermolecular
addition,
various
growth
space
are
perfectly
the
found
in
a
Piez,
there
structure
appropriate
than be
to
and
and
but
for
mineralized
that
(Hulmes
(Trus
distances
conform
Therefore,
the
arrangement
sufficient
(1972)
as
arrangement
important
m i n e r a l i z a t i o n may
steric
a l . , 1985).
such
three-dimensional
and
mineralizing
that
do
biologically the
et
three-dimensional
formation
that
specific.
The
there
the
over
which
pattern.
crystal
packing
microfibril
regular
for
(Hulmes
arrangements
molecular
or
be
significant
structure
are
greater
tissues
certain tissues
the
geometry
of
packing
and
result in different
may
and for be
a l . , 1987).
Crosslinkinq Covalent important connective specific
c r o s s l i n k s between
determinants tissue
of
matrices
the and
collagen
mechanical are
an
molecules properties
example
p o s t - t r a n s l a t i o n a l heterogeneity
of
of
the
type
I
are of tissue
20
collagen.
Crosslinking
aldehydes, peptides,
i n t h e amino with
amino
i n the t r i p l e
et
1986).
demonstrated
Two
sclera
and r a t t a i l
most
tendons
labile
(Eyre,
(Tanzer
reducible
Glimcher, crosslinks
1973).
o f bone
1974).
collagen
These
crosslinks
pyridinoline chemistry
undergo
(Fujii
i n type
and T a n z e r ,
I collagen
residues
from
o t h e r c h a i n s form
Although
i ti s believed
involved.
Banes
can r e a r r a n g e t o ( E y r e and reduced
chains,
that
ages
The
crosslinking
initially
involves
but subsequently,
t r i and t e t r a - c h a i n
the crosslinks
as t o the nature
e t a l . (1983)
bone
to nonreducible
and n o n - m i n e r a l i z e d c o l l a g e n debate
(aldimine)
As n o r m a l
1974).
fibrils
two c o l l a g e n
some
participate in
The two p r i n c i p a l
a transition
from
remains
l i g a m e n t and
are dihydroxylysinonorleucine
residues
mineralized
cornea,
collagen, the
crosslinks
(DHLNL) and h y d r o x y l y s i n o n o r l e u c i n e . DHLNL
on
on h y d r o x y l y s i n e
also
I n bone
1971).
been
skin,
i n bone, may
(Yamauchi
- one b a s e d
are Schiff-base
ketoamine
1972; Mechanic,
have
i n adult
Histidine
et a l . ,
stable
hydroxylysine
the o t h e r based
crosslinks
specific
nonhelical
linking
collagens
predominates
1987).
and
of
regions of molecules
of cross
tendon;
et a l . ,
(Mechanic
terminal
predominates
which
reducible
compounds form
which
pathway
crosslinks
helical
f o r the f i b r i l l a r
aldehydes
the reaction
of lysine
pathways
lysine
aldehyde
from
and c a r b o x y
groups
located al.,
results
links.
i n the
are different
there
of the structures
d i d not find
pyridinoline i n
21
the
mineralized
collagen
o f bone
non-mineralized
portion.
They
physically
inhibits
approximation found for
that
suggested
of the collagen particle
resulted
m o l e c u l e s . Wu size
i n protease
found
used
They
mineralized
and n o n - m i n e r a l i z e d t i s s u e . t o b e o f some
process.
At t h i s
further
clarification.
Biological
having
time
apatite
(Jackson
et a l . ,
rod-like
structure
field field
difficulties a wide
measurements a mean
Robinson et
(Myers
dark
characteristic
given
al.,
thin,
1978; Weiner
bright
selected-area
Direct
of
(1 9 8 8 ) bone
pyridinoline
i n pyridinoline I
between
expect
in mineralization
there remains
crystals
size
a need f o r
1978; L a n d i s
been
and Traub,
have
been
electron electron
range
1965).
measured
microscopy microscopy with
field
by
x-ray
and (SADF).
Due t o
this
been r e p o r t e d . microscopy
have
(Robinson, 1952;
1952; S t e v e - B o c c i a r e l l i , and G l i m c h e r ,
shape
The
studying
electron
3D t o 70nm
either
1986) o r as a
o f v a l u e s have
by b r i g h t
described
elongated, plate-like
associated
between
and Watson,
have
and Engstrom,
of the c r y s t a l s
diffraction,
technical
by p r e v e n t i n g
i n powdering
significance
though,
an i r r e g u l a r ,
dimensions
mineralization
Apatite
Individual as
no d i f f e r e n c e
i n the
and Eyre
digestion
crosslinks.
crosslinking
that
formation of p y r i d i n o l i n e
the small
analysis
b u t i t was f o u n d
1970; Jackson
1978; Weiner
and
Price,
22
1986).
However,
electron
visual
micrographs
background
electron
differences Hunziker,
interpretation
results density
i n detection
1988).
interpretation
of c r y s t a l s
crystals.
Second,
position
leading
to larger
small
section
crystal
contains
probability the
X-ray from
that
t o 36nm
Engstrbm,
1985;
Grynpas small
which
crystals.
the cumulative crystal
(Myers
has been
reported
even
size.
(Arsenault
the thinnest the
density
o f many
and Grynpas, forcrystal
i n the l i t e r a t u r e as
et a l . ,
et a l . ,
and Grynpas,
to i t s large
1988).
length
Jackson
e t a l . , 1983; Boskey
i s related
Due t o
e t a l . , 1989) and
electron
results
more
in crystal
increases
e t a l . , 1986; Arsenault size
biased
of larger,
and Engstrb'm, 1 965;
1972; Bonar
crystal
a
and
of c r y s t a l
This
First,
results i n
be o v e r l a p
(Hunziker
d i f f r a c t i o n has produced
1978;
The
many
problems.
expect
cross-sectional size,
i s a single
1 Dnm
may
estimates
of overprojection
perception
crystals
one w o u l d
there
field
(Arsenault
due t o s e l e c t i o n
obvious
the
i n two b a s i c
of the specimen
Therefore,
of size
of bright
1988).
surface
area
between
2 85-265m 1963;
/g ( R o b i n s o n
Miller,
Grynpas, extremely and
may
1 9 5 2 ; Hodge a n d
and P r i c e ,
The b i o l o g i c a l
important
Studies size
1984; Weiner
1988).
crystal
and Watson,
control
i n determining
Petruska,
1986; A r s e n a u l t of c r y s t a l
biomechanical
and
size i s properties
chemistry. which
mislead
report
the reader
single into
mean
values
assuming
that
for crystal there
i s a
23
narrow
range
standard
i n the
sample.
deviations
reported
heterogeneous
population.
of
this
range
all
of
large the
maturity. on
the
stage
depend are to
on
of
the
emphasize the
(Arsenault crystals
apatite
for
literature,
preparation associated Arsenault
leads
to
apatite electron
gap
s i z e and
1988).
at
that
would
the
For
not
of
times
crystals
seem
and
like to
Petruska
collagen
example,
l e a s t ten
also
I would
Hodge
packing
depend
I t may
adjacent
do
in
tissue
remodelling.
in
two-dimensional
are
of
shape
dimensions
zone
sources
common p r o b l e m
different levels.
crystal
the
a
possible
standardization
such
large
the
the
width
width
of
of
the
the
zones. microscopic
major i t has
ultrastructure
aqueous
the
at
A
the
for
several
sizes.
crystal
Grynpas,
electron pose
evidence are
environment
s i z e of
gap
There
i s the
growth
measured
predicted In
in
and
are
contrary,
m i n e r a l i z a t i o n and
that
(1963) model
the
crystal
that
local
restricted
reflect
of
techniques I assume
On
are
and with
and
stains
the
c h a r a c t e r i s t i c s of
t e c h n i c a l problems.
Often,
been
changes
a
suggested
r e s u l t of
specimen the
totally
ambiguities c r y s t a l s are
that
the
methods
visualization.
solubility
Hunziker
microscopy
studies
(1988)
showed
demineralize
i n what labile
i s being to
(Arsenault,
of
problem
is
conventional sections
stained.
As
tissue
apatite.
r a d i a t i o n damage 1988).
mineral
for
such
that thin
the
in
used
One
properties
in
a
and
In
this
addition,
during
consequence
of
24
these
technical
difficulties,
methods
quick-freezing/freeze-substitution 1988;
Arsenault
(Landis
et
developed
to
differences
there
may
between
model
the
be
calcium
two.
biological structure
and
been
the
analyses
ideal
carbonate
(Hagen,
These
stages
of
geological
or
reveal
result
synthetic
Second,
relationships in
other
may
mineralization. must
that
First,
1973).
arrangements
crystal structure
some
fluoride,
stoichiometric which
are
formula.
and
phosphorus
considered
there
including
various
Hunziker,
techniques
have
i n bone
alternative
apatite of
Chemical
from
arrangements.
at
a l ; 1980)
exist
chloride, also
important
anhydrous
for biological apatite
between
crystalline
( A r s e n a u l t and
h y d r o x y a p a t i t e , Ca^^(P0^)gOH, 4s
impurities
magnesium,
et
as
a r t i f a c t u a l changes.
differs considerably
numerous
and
Landis
minimize
idealized
bone
a l . , 1988)
a l . , 1977;
Although the
et
such
be Therefore,
deviate
from
hydroxyapatite
the
of
ideal
composition. The crystal the been
unit
symmetry
length the
and
described
c.
of
structure.
apatite, and
cell
the
The of
as
unit
apatite
I t has
chemical a
cell
positions
axes, of
the
a l l the
elements,
i s defined
their
are
of
the
by
three
axes
labelled according
The
unit
cell
the including
c r y s t a l and
(Hagen,
i n c l i n a t i o n to
atoms.
throughout
characteristics,
s t r u c t u r a l molecule
c r y s t a l planes the
i s repeated
one of
1973).
In
labelled to
another, the
has
a,b
the and
crystal in
25
calcified
turkey
a=Q.942nm
a n d c=0.688nm
basic
cells
consistent of
tendon
of c r y s t a l
i s hexagonal (Myers
diffracted electrons
selected-area
dark
Apatite/Collagen Biological
apatite
the non-collagenous
their
(Glimcher,
preferred
within
the
in a
visualization
crystal
planes i n
microscopy.
the collagen
and Traub,
of the c r y s t a l s within
suggested
location
that
t h e gap zone
f o r mineral
1986).
(1963)
and
Yet the
remains the
be t h e
within
fibrils
because
of the p o t e n t i a l space
a v a i l a b l e . Since
that
certain
studies
this
et a l . ,
1977).
localized
overlap
localization focussed result
zone.
view
(1983)
i n t h e gap zone
The p r i m a r y
recent
arrangement
information
deposition
believed
are within
both
that
i s
throughout
f o r the s t i l l
remains
t o e x i s t as a
of collagen
suggests
time,
the mineral
but spreads
o f c r y s t a l s i n t h e gap zone
of the staggered
(White
states
explanation
on t h e p o t e n t i a l s p a c e
However, mineral
supported
A l t e r n a t i v e l y , Glimcher
initially the
have
the
i n proposing
would
deposition
leg
outside
the f i b r i l s
Hodge and P e t r u s k a
turkey
fibrils
e x t r a c e l l u l a r matrix
1959; Weiner
of debate.
model
These
electrons
crystals i n mineralizing
within
subject
1965).
Relationships
and bone a r e f o u n d
localization
specific
electron
tendon
fibrils
diffract
makes p o s s i b l e
from
field
the dimensions
and Engstrom,
organization
manner and t h i s
with
early
molecules. sites
t h e gap and o v e r l a p
of zones
26
(Arsenault, of
mineral
overlap
1 988).
is initially
zone
progresses. the
but
this
a
previously The
to
of
specifically
the
viewed
by
observed
alternate
to
This
arrangement
of
the
axial
i s most of
the
axial
Conclusion
to
arrangement
proportion
zone
as
the
than
in
the
mineralization overlap
collagen
explanation
of
space
or
crystal in
than
zone
then
fibril has
been
the due
dark
collagen
molecules
planes
run
are
electron
crystals is
length
the
to
(Engstrom
field
apatite
fibril to
tendon
crystal
o r i e n t a t i o n of along
i s considered
turkey
selected-area
likely
acid
(Arsenault,
three-dimensional such
that
the
structural
rotation
properties
the
because spaces.
of
the
fibrils of
collagen
localization
First,
collagen
negative
Review
sequence
and
reasons.
intrafibrillar positive
the
When a p a t i t e
Historical
amino
several
potential
in
the
fibril.
biomineralization for
gap
equal
orientation reflects
collagen
The
within
apatite
1951).
the
apatite
the
more
molecules
microscopy,
of
in
is localized
complex
collagen
Zetterstrom,
1988).
reported
described.
c-axis
parallel
greater
apatite
much m o r e
(1 9 8 9 )
becomes
If apatite
i n t e g r a t i o n of
requires
and
Arsenault
e f f e c t s on
of
play
mineral
a
may
result in
the
amino
nucleation
in
packing
regions
flexibility
specific
role
apatite
three-dimensional
regional
Second,
may
of
or
acids or
may
have
growth
of
27
apatite such
crystals.
Thirdly,
as h y d r o x y l a t i o n ,
factors
which
specific
locations
mineralization literature.
where
collagen
fibril
amino
apatite? the
and t h e r o l e
acid
D period? sequence
Are there
mineralization
acid
of type
some
precise
t h e mechanisms of e x i s t s i n the
unanswered. F o r
i n relation
to the
study
between
and t h e l o c a t i o n o f which
i n the overlap
the following
of
sequence.
of collagen
of these
be
Fourth,
process
i s the r e l a t i o n s h i p
of collagen
of apatite
at
remain
located
What
in-the
I collagen
questions
properties
to understand
collagen
regarding
i s the mineral
localization
attempt
basic
of a p a t i t e .
involved
by t h e a m i n o
uncertainty
Numerous
example,
proteins
i n t e r a c t with
determined
Considerable
the
the l o c a l i z a t i o n
non-collagenous may
modifications
c r o s s l i n k i n g a n d g l y c o s y l a t i o n may
determine
mineralization
post-translational
aspects
zone?
can
explain
I n an
of
was c a r r i e d o u t .
28
INTRODUCTION
Type numerous
I collagen connective
mineralization collagen formed al.,
molecule
portion
terminal, that
organized rise
o f bone,
1963; Ueis,
showed
tissues
i n a regular
adjacent
ends
and each
neighbor
by 67nm,
there
for
(1963)
are l o n g i t u d i n a l l y fashion
which
separation
The r e p e a t i n g
was e n v i s i o n e d overlap
region, where
molecules. would
there
between
would
lateral
residues
of the
two r e g i o n s ,
i n length,
a
and a "gap"
be p o t e n t i a l
space
between l o n g i t u d i n a l l y
Hodge and P e t r u s k a
(1963)
suggested
that
be t h e p r e f e r e n t i a l l o c a t i o n o f a p a t i t e
mineralization
growth.
D period
as having .4D
gives
are linearly
acid
the r e s u l t of the separation
crystal
Hodge and P e t r u s k a
t o 234 amino
.60 i n l e n g t h ,
during
N- a n d
corresponding
region,
crystals
triple
to i t s
et a l . , 1973).
gap r e g i o n
a
(Piez et
and s m a l l
The m o l e c u l e s i s a 35nm
chains
i s staggered
packed
arranged
have
The
i n length
molecule
densely
the
molecules
i n the
l e g tendons.
residues)
67nm, D - s t a g g e r e d
period.
that
acid
to
involved
peptide
molecules
portions.
the collagen
such
as
These
(1014 amino
nonhelical
fibril
and t u r k e y
one a 2 ( l )
1985a).
arranged
collagen
and i s d i r e c t l y
i s a s e m i f l e x i b l e r o d , 300nm
to i t s axial
(Hulmes
structural integrity
dentin
by t w o a 1 ( I ) a n d
helical C-
provides
due t o t h e s p a c e
available
29 The results
regular
i n the periodic
specific 1952). amino
D-stagger
Meek acid
e t a l . (1979) were sequence,
of type
individual
amino
coefficient
I collagen acids
density
of their
amino
Hulmes,
approach mineral
1 9 8 4 ; Meek
into
collagen
relative
present
study,
density
analyzed
and compared
have
used t h e
with
specific 1982b; An
Chapman
the impregnation
amino
acid
t o heavy
of
of apatite
composition. metal
and d i f f r a c t e d e l e c t r o n s t o models
classes
analogous
by t h e l o c a l i a t i o n
i n addition
electron
correlation
and t h e t h e o r e t i c a l
and Chapman, 1 9 8 5 ) .
t o t h e known
of
the e l e c t r o n - o p t i c a l
e t a l . , 1982a,
fibrils
and t h e s t a i n i n g
to determine the
stains
to understand
(Bear,
t h e known
A high
studies
of collagen
(Tzaphilidou
c a n be u s e d
crystals this
data
with
the l o c a l i z a t i o n
collagen
of electron-dense
acids
to integrate
between
Subsequent
also
acids
implications
the f i b r i l .
found
stained
model.
sequence
localization
and
o f amino
t o model
within
( r = . 9 2 B ) was
of positively
acid
able
molecules
structural information
density
of
localization
s t r u c t u r a l and h i s t o c h e m i c a l
patterns
amino
of the collagen
In
s t a i n i n g , the
of apatite are
of collagen
amino
acid
sequences. The
role
mineralization suggestion reported exclusion
and i n t e r a c t i o n s o f type process
remains
an e n i g m a .
by Hodge a n d P e t r u s k a the occurrence from
(1963)
of apatite
the overlap
zone
I collagen
Corroborating
several
authors
i n t h e gap zone
(White
i n the the have
and i t s
e t a l . , 1977;
30
Berthet-Colominas initial
localization
zone
followed
zone
(Glimcher,
earliest the
1983).
zone
and t h a t
overlap 1989)
zones
. There
collagen
remain
an i n t e r m e d i a r y
tissue
proper
collagen
analyses
individual The
present
acid
the
the
proceeds
(Arsenault,
the role of
between
apatite
collagen
and
an i n t e r a c t i v e p r o c e s s
tendon
thereby,
force?
Many
instead
high
or
studies
o f bone
degree of
permitting
of the m i n e r a l i z a t i o n
collagen
was u n d e r t a k e n
localization of type
of apatite
t o be r e l a t e d
inverse
i n t h e gap and
of b i o l o g i c a l
of the tendon's
study
sequence
organization found
within
ease of
process
along
fibrils.
intrafibrillar amino
turkey
that the
(Arsenault,
concerning
i s the dominant
alignment,
the
overlap
indicates
gap zone
of mineral
association,
because
fibril
sequential
to'the
questions
the mineralized
the
crystals are localized
the association
whether
used
throughout
i n t h e mechanism
i s a passive
describe
t o be e x c l u s i v e l y i n t h e gap
as m i n e r a l i z a t i o n
many
whether
authors
information
the proportion
mineral
have
Recent
apatite
changes
Other
spread
i n addition
structure
deposition-
of mineral
by p r o g r e s s i v e
detectable
overlap
1988)
et a l . , 1979).
fibril
localization
of apatite
in relation
I collagen.
within
The
the collagen
to the polarized as determined
r e l a t i o n s h i p between
to investigate the
asymmetric D period
t o C-
was
d i r e c t i o n of
by t h e s t a i n i n g p a t t e r n .
the l o c a l i z a t i o n
of hydrophobic
to the
amino
acids
of apatite
was f o u n d
A
and
t o be
31
evident
i n both
conclusion effect, serves
of
which to
this
determined
by
controlled
influences.
gap
and
thesis
o v e r l a p zones.
therefore,
i s important to
influence
distribution
be
the
of
the
the by
the
crystals
gap other
region
i s that
collagen
distribution does
not
primary
the
hydrophobic
structure,
of a p a t i t e . appear
i n collagen
c o l l a g e n o u s or
The
to
fibrils
also The
be
solely
but
non-collagenous
could
32
MATERIALS
Tissue
Preparation
Small tendons
of
solution 0.05M then
10
of
f o r 15
sodium
of
old
1%
pH
changes each
w/v
osmium
7.4, of
followed placed 100%
water by
in a
3
2%
w/v
f o r 2h.
0.05M
followed
by
1h
was
(50%,
70%, of
s e r i e s of
r e s i n ) f o r one
sucrose
resin
i t was
(gold
i n t e r f e r e n c e c o l o r ) were
mesh
or
with
Reynolds UA
200
polymerized
mesh
i n 75%
lead
glycerol under
f o r 2h.
these
pH
portions
turkeys
60
C
were
These
of
for leg
excised
Non-mineralized
c o n d i t i o n s and
f o r 16
so
methanol
in
f o r 15
(3:1,
Thin
min
each
then 1:1,
1:3,
of
100%
sections
formvar-coated
1963)
75
stained
f o r 6 min
and
3%
min.
tendons and
of
from
placed
tendon
areas
0.05M
placing
s e c t i o n s were
6
a
overnight
h.
on
(Reynolds, 4.2
by
2%
and in
and
f r e s h change
placed
grids.
citrate
methanol
Mineralized domestic
copper
at
a
out
resin
Following
tissues with
t i s s u e s were
cacodylate
of
a
and
t i s s u e s were
acetone:Spurr's each.
in
sucrose
3x100%)
The
leg
post-fixation
carried
90%,
acetone.
hour
the
80%,
placed
The
sodium
t e t r o x i d e , 2%
Dehydration
changes
i n f i l t r a t i o n ' of
old
were
tissues in increasing concentrations
distilled
w/v
non-mineralized
turkeys
g l u t a r a l d e h y de,
min
cacodylate.
excised,
domestic
cacodylate, 3
METHODS
Visualization
freshly
v/v
rinsed with
solution
of
week
2.3%
sodium
sucrose
the
pieces
and
AND
10 in
becomes interest
to
14
week
100% transparent near
the
33
mineralization mineralized
front,
regions
glycerinated
unmineralized
c a n be
tendon
were
tendon
identified.
placed
( 3 : 1 , 1:1,
100%
3 changes of acetone
tissues
were
then
Sections tendons
with
depending
on
were
collagen
banding
selected-area
were
dark
water.
75 m e s h
electron
collagen
on
these
the
uranyl
molecular
their
banding
o r 600 field
min
analysis on
stained
with
were
1%
o r i e n t a t i o n of collagen and
to maintain
to
In order fibrils the
bright
were
and
f o r 10 m i n .
1988).
and
aqueous
f o r 15 m i n
found
on
visualize
produce the conventional they
on
grids.
sections to
and
color
microscopy
In order
of
distilled
placed
copper
of mineralized
(Arsenault,
patterns
t o be e m p l o y e d .
quickly
4.2
the
localization
electron
3.2
The
thicknesses
color
hexagonal
were
each.
along
interference
were
( U A ) a t pH
I collagen
l e g tendons
gray
( P T A ) a t pH
acetate
of type
3 changes of
microscopic
of mineral
sections
sections
acid
technique
sections.
s t a i n i n g methods
pattern turkey
dark
unstained
banding,
by
of
r e s i n as above.
interference
were
microscopy
phosphotungstic aqueous
field
These
Selected-area
performed
gold
of
series
f o r 15
in different
for electron
for analysis
formvar-coated
field
knife
stained
Sections
followed
i n Spurr's
the v i s u a l i z a t i o n
t o be
distilled
1:3)
heavily
pieces
cut i n l o n g i t u d i n a l planes
a diamond
Sections
water.
embedded
Small
i n a graded
glycerol:methanol m e t h a n o l and
and
1%
Although
banding demineralize to by
native
visualize analysis
of
34
distribution sections Bright using The
the
field
stained
300 e l e c t r o n
o f SADF
beam
microscope
positioned
from
This
selected
In this
collect
planes
electrons
of apatite
technique
the objective
specific
study
a t 80 k V .
(Jackson uses
aperture.
tilting
correspond
of
are
With the beam
c a n be c o l l e c t e d
diffraction
pattern
aperture
d i f f r a c t e d from
et
mode t h e t i l t e d
electrons
the objective
which
performed
diffracted electrons
the c h a r a c t e r i s t i c electron
apatite.
f o r 5 min.
operating
elsewhere
i n the d i f f r a c t i o n
so t h a t
mineralized
a n d SADF w e r e
microscope
1988).
through
and
i n 100% methanol
i s described
so t h a t
to pass
electron
1 % UA
microscopy
1978; A r s e n a u l t , main
non-mineralized
with
electron
a Philips
allowed
to
were
technique
al.,
is
of apatite,
was
of
positioned
the c - a x i a l
lattice
to the (002) d
lattice
spacing.
Computer The
Modelling system
of modelling
fibrils
used
by
et a l . (1979).
Meek
(Highberger chick
here
each
collagen
i s an a d a p t a t i o n The amino
acid
were
translated
as a s i n g l e
acid
sequences
were
turkey
i s unavailable
either
turkey
or chick
into
string used
data
into
because
at this
sequence
i n collagen
of the system
e t a l . , 1982) a n d a 2 ( l )
collagen
entered
amino
time.
sequences
(Boedtker single a text amino
developed for a l ( l )
e t a l . , 1985)
letter
codes and
editor. acid
Chick
data f o r
The r e s u l t s when
are expected
to d i f f e r
using
only
35
minimally homology single amino
because there between
positions to
by
fifty
regions
acids
1984).
stacked
to simulate
four
were
i n width.
deviate
then
identified.
amino
pattern
of collagen
abbreviations
spaces. and
then
size
D,E,R,K
of electron
determined
reduced
of collagen by f o u r
from
classes
p. i v ) were acids
were
i m a g e s . The
stained
with
a l t e r n a t i v e methods
amino
acids
(K,R a n d H ) ; 3) w i t h collagen;
et a l . (1982b).
their
o f amino the
i n their
replaced
by
by c o m p u t e r
to the equivalent molecular
of the d i s t r i b u t i o n
charged
Tzaphilidou
and
of comparison:
(D a n d E ) ; 2 ) w i t h
stained
because
a l c o h o l i c UA w a s
acids
PTA/UA
left
was r e d u c e d
amino
aqueous
i n length
to represent
charged
of
repeating
linearity
photographically
a t h e o r e t i c a l model
assumed
and H ( s e e t a b l e of
amino
microscopic
were
f o r the positive staining
The r e s u l t a n t d i s t r i b u t i o n further
relative
D units
c a n be e x p e c t e d
In order
acids,
a l l other
The
The
i n the l o c a l i z a t i o n
Functional
responsible
f o r amino
while
orientation
with
acids
error
the greatest
a r e n o t known.
charged
positions
Some
i n the telopeptides
conformations
chains
and Hulmes,
molecules
these
2(l)
a
These
D u n i t s , 234
i n width.
(Chapman
then
acids
repeating
and 5 m o l e c u l e s a
amino
et a l . , 1985).
into
o f t h e two 1 ( I ) and t h e
were
degree of sequence
(Boedtker
organized
i n length
be a 1 - a 2 - a 1
units
species
s t r i n g s were acids
i s a large
1)
of negatively
a model
of
a scaled
and 4) w i t h
positively micrograph
data
The p e r i o d i c d i s t r i b u t i o n
from of the
36
hydrophobic and
amino
I , has been
structure acids
acids
considered
(Hofmann
were
having
long
t o be
e t a l . , 1978)
selected
and
then
side-chains,
important
reduced
to the scale
collagen
fibrils
for direct
The
contrast
of t h i s
image
hydrophobic groups
as w h i t e .
considered helix
groups
t o be
stability
as b l a c k
and
Hydroxyproline important were
also
represents
areas and
high
amino of
comparisons. areas
which
flexibility
in this
\J, L
low i n
i n hydrophobic
praline,
i n molecular selected
visual
M,
fibril
These hydrophobic
mineralized reverse
in
F,
fashion.
are and
37 RESULTS
Electron
microscopic
glutaraldehyde tetroxide
this
an e f f i c i e n t
a r e seen
cell The
system
Tenocytes
extracellular
because
t o have
the section
Intracellular
Rough
f o r studying
These
extend
features of these
i n a parallel fibrils
microns.
Their
70nm
alternating
bodies
axial light
separated
of c e l l s
a r e about
the c e l l
fibrils.
i n width. vary
and i t s p r o c e s s e s . secretory
and m i c r o t u b u l e s a r e
matrix,
appear
collagen f i b r i l s the c e l l s .
t o vary
from
are
Individual f o r many
approximately
low m a g n i f i c a t i o n t h e
of these
and dark
through
i n the tenocytes
c a n be f o l l o w e d l o n g i t u d i n a l l y
repeat
diameters
and
60nm
by
elongated
to collagen
f a s h i o n between
diameters
makes
cells.
t o 300nm. A t a r e l a t i v e l y
pronounced
columns
of cross-sectional
structures,
the extracellular
collagen
(fibroblast-like
reticulum, mitochondria,
prominent
clearly
are generally
parallel
and between
filamentous
aligned
cells
processes
vacuoles,
osmium
the mineralization
into
organelles visualized
endoplasmic
with
( F i g u r e s 2a and b) w h i c h
i s through
which
to cell
Within
post-fixed
and l e a d c i t r a t e ,
a variety
smallest of these
cell
UA
are organized
matrix.
processes
from
tendon,
of non-mineralized,
the organization of tenocytes
and c o l l a g e n f i b r i l s
process.
and
turkey
and s t a i n e d w i t h
illustrates cells)
fixed
examination
fibrils
regions
which
consists
of
a r e due t o t h e
38
Figure
2a a n d b. E l e c t r o n
tendon.
Parallel
processes (F).
columns o f t e n o c y t e s (T) and
endoplasmic
reticulum
and
microtubules (arrow)
are
commonly
observed
extend
analysis tetroxide
of non-mineralized
( C P ) a r e s e p a r a t e d by p a r a l l e l
Rough
fibrils
micrographs
along
longitudinally
fixed,
b=2DD; a X 2 5 . 0 0 0 ;
cells.
X50,000.
citrate
fibrils
m i t o c h o n d r i a (ffl), organelles
The p a r a l l e l
f o r many
fibrils.
UA a n d l e a d b
collagen
are intracellular
i n these
individual
(rER),
their
microns
which
collagen
permitting
Glutaraldehyde/osmium stained.
Bars
a=400nm,
40
specific
staining
molecules
within
magnification composed been
1960).
an
Each
intensity, The
fibrils which
i n Figure
a^»
and
not
t o be
are
very
a^)
single
(e^
62)
and
molecular the
band
a^
c^
with
well
defined
lines
other
distance separating
borders;
tend
(b^ and have
a
b^) well
apparent
i s an the "e"
separated
a r e due
the l i g h t
s t u d i e s have
which
bands
f r o m ea'ch
a narrow l u c e n t space.
stained regions
d are within
PTA/UA
" c " bands; f o r
"d" band
narrowly by
used;
i s readily
the s i n g l e
are separated
c and
they
and
are f a i n t ;
these
morphological
bands
" a " and
only
bands.
" a " bands ( a ^ ,
the adjacent c^)
of
spaced bands
separated,
have
staining
aqueous The
The
also
between
level
follows:
and
3 the densely
b, w h i l e
to each band.
be
Schmitt,
to other
stained with
"b"
well
and
methods
d e l i n e a t e d ; t h e two
are d i s t i n c t
and
been
a r e as
( c ^ , C2>
intense
Figure
have
to
which
structure,
the s t a i n i n g
are narrow, c l o s e l y
the others
other,
i s shown
i t srelative
therefore the f i n e
bands
distinct border
by
collagen
higher
repeat
relationships
definition
clearly
" c " bands
and
spatial
3a
clear
a^,
while
At a
of narrow bands
identified
d e p e n d i n g upon
of these
the
and
and
details
defined
c a n be
width
gives
each a x i a l
ordered
" a , b , c , d , e " n o m e n c l a t u r e (Hodge
band
varies
structure.
asymmetric series
intensity,
bands
the f i b r i l
( F i g u r e 3a)
o f an
given
p r o p e r t i e s of the highly
shown
t o bands
r e g i o n . At that
t h e gap
e,
In a
the zone,
-
41
Figure
3 a . The p e r i o d i c
fibrils. to
The D r e p e a t i n g
the recognizable
intensity c,
staining pattern
and t h e i r
d , a n d e" p e r m i t
unit,
series
defined
the determination
displayed
image
display
characteristic resulting the
bands
N- t o C-
displayed collagen both PTA
of p o s i t i v e l y
image
a n d UA
alignments
the s p a t i a l of these
a r e l a b e l l e d from
d i r e c t i o n i s from
image
images
depicts
from
Figure
of Figure
fibril.
and n e g a t i v e l y
i s s h o w n i n t h e same m o l e c u l a r
This
charged
distribution
charged
amino
amino Figure
and acids; the
"e t o a " i n d i c a t i n g
left
to right.
3b i s s c a l e d
3a ( i n s e t ) .
Bar=10Qnm;
" a , b,
3b. A computer
o r i e n t a t i o n as
X13D,QQ0.
that
A computer
and a l i g n e d
The b a n d i n g
l i ei n r e g i s t r a t i o n . Glycerol stained;
their
o f t h e N- t o C-
o r i e n t a t i o n of the collagen
3a.
by
according
r e l a t i o n s h i p s . The b a n d s
molecular
acids
collagen
( D ) c a n be l a b e l l e d
of bands
spatial
of
to the
patterns
treated,
aqueous
of
42
3b
43
longitudinally a., _ . , e„ of
C2
^» The
. , d and
b2
^ and
banding
distribution therefore, known
align
and
acid
c, while
a^
(Hodge
collagen
permit
unambiguous
The
t o C-
with
asymmetric
pattern found
of bands that
scaled 3a
a"
and
fibrils
or of opposite
derived and
orientation. orientation of
insert).
i n turkey
o r i e n t a t i o n and
orientation within
to the a n a l y s i s
and
image
presentation 3b
of
and
of the bands
molecular
the
acids
pattern
(Figure
of collagen
acids;
from
amino
to the stained
(Figure
adjacent
of molecular
i s critical
c, b,
then
the
In our
banding
was
intensity
determination
of amino
charged
fibril
correspond
t h e " e , d,
o f t h e same
determination
3b)
d i r e c t i o n of collagen
correlates
i t was
and
represents
I collagen.
model
collagen
registration
computer modelling
fibril
This
consists
constructed
t h e " a t o e"
fibrils.
The
be
(Figure
to give
groups
c a n be
of
1963).
I collagen
sequence of type model
consists
zone
Petruska,
of charged
inset).
may
and
density
to the stained
study
the overlap
of type
aligned
by
molecules
pattern
generated
vertically
native
collagen
t h e o r e t i c a l models
amino
computer
arranged
of a p a t i t e
the
this In
this
tendon thus,
the
collagen
localization.
44
Figure
4. Low m a g n i f i c a t i o n o f g l y c e r o l
distal
to the mineralization front.
appear
white
translucent. (Min).
the
dense,
least
regions
stages
i n unstained
mineral
density
of c a l c i f i c a t i o n
(arrow)
proportion of mineralized matrix
more
c
are evident
regions
increases
within the collagen
of non-mineralized
heavily mineralized region
X5Q,0Q0.
localized,
electron-lucent, non-mineralized
(arrowheads)
subsequent
m i n e r a l i z a t i o n was stages
calcification.
electron within
matrix.
5b.
and p e r i o d i c
fibrils
matrix
but w i l l
Bars=200nm;
near
s e c t i o n s . 5a I n
small
The
Small
proximal
of m i n e r a l i z a t i o n present
otherwise
5c.
to
a n a l y s i s of early
the
banding
are
d e p o s i t i o n . Bar=0.5mm; X 2 5 .
5. D i f f e r i n g
with
distal
just
regions
regions
of visible
f o r electron microscopic
mineralization front
regions
precedes
a t the boundary
apatite crystal
Figure
The c a l c i f i e d
the non-mineralized
Mineralization
Tissue
selected of
while
t r e a t e d tendon
i s present.
(*) r e m a i n
be f i l l e d
a X45,000, b
in a
i n by X40,QQQ,
47
Treatment with
of mineralized
g l y c e r o l makes
regions
collagen
easily recognizable
insertion
of the tendons
mineralized. decreases
The d e g r e e
away
from
domestic
translucent (Figure
into
which
was
for electron
microscope the of
electron
dense
the
matrix
which
do n o t h a v e
which
pattern
which
mineral
At sections
heavily
remains.
progressive
occurs
D period
within
exibits
with
nature
there
In t h i s
of mineral,
magnification
at this
seemingly
unstained,
at the mineralization
of
are
study
front
regions
I am
pattern
i s most l i k e l y
(Figure
of
the
e x t r a c e l l u l a r matrix.
feature
gradient
5b). In addition
a periodic
deposition
at
a periodicity
(Figure
pattern.
mineralized
The main
spread
higher
of the
regions
5 a ) . As t h e d e g r e e
has a p e r i o d i c
the non-collagenous a r e more
analysis
localized
are evident
banding
a banding
mineral
non-periodic
within
region
i t an a p p a r e n t
small,
(Figure
to the collagen
considering
of the
to
longitudinal section
has w i t h i n
Initially
increases
the mineral
which
i t disappears
microscopic
unstained,
calcification
electron-lucent
to
front
spread.
equivalent
progressively
i s representative
an i n d i v i d u a l ,
calcification
are heavily
f r o n t . When v i s u a l i z e d i n t h e e l e c t r o n
mineralization mineral
mineralized
to the
the tendons
the i n s e r t i o n u n t i l this
mineralization
and t h e
of m i n e r a l i z a t i o n
observation;
l e g tendons
4 ) . Near
bone,
gross
prepared
turkey
only
because occurring
In
regions
5c) the banding stage
i s the
i n a l l dimensions. longitudinal
of turkey
l e g tendons
48
(Figure
6a)
electron
reveal
density
a distinct
of a p a t i t e
non-mineralized
regions
demonstrate
structural
any
mineralization, zone; the
however,
overlap
zone
has
random
most
zone.
on
More
The
side,
the
lucent
area.
occurs
i n the mid-portion.
in
contrast
marked.
detail.
Within
similar between
by
occurs
zone
present that
neither
appears
to
be
zone
gap
gap
is
the
the
i n the
electron-lucent
In r e g i o n s
early of
are
observed
and
overlap
the
early
which
asymmetrically
features
of
observes
i s a single overlap
to
not
i s also
density
an
do
In regions
one
due
contrast,
section
material
gap
pattern
In
density
of e l e c t r o n
either zone
same
specifically,
within
deposition
gap
the
electron
homogeneous.
delineated,
crystals.
electron-dense
a pattern
nor
of
repeating
line
in each
and
positioned
mineralization
increased but zones
the
mineral difference
i s less
49
Figure
6.
The
bright
field
localization image
mineralized
turkey
pattern
to
due
compared
to
a
less
(OZ).
The
zones
demarcated
gap
zones
space 6b
and
c.
unstained of
has by
a
bordered arrows).
Two
collagen
the
mineral
content
i n gap
an
i n the
lucent
space
(white
both
sides
to
is identified and
by
Non-mineralized
as
white
overlap
the
lengths)
bright field
(GZ)
overlap
zones
within
Also,
narrow
lucent
of
of
which
X120,000.
the
matrix
(*).
mineralized
specific
at
gap This
fibril.
localization
appear
in
the
micrographs.
h i g h l i g h t s w i t h i n both (OZ)
as
arrowheads).
portion
electron
zones
m i n e r a l i z a t i o n . Bars=100nm;
a
images
showing
(c-axial
Apatite
(GZ)
on
given
of
banding
zones
distribution
A
section
inherent
interspaced
asymmetric
fibrils
contrast
of
longitudinal
showing
r e p r e s e n t a t i v e SADF
apatite crystals
zones
unstained,
tendon
mineral
reverse
gap
an
a p a t i t e i n c o l l a g e n . 6a.
i s consistent within a
are
(white
leg
high
mineral
distribution
of
of
the
earliest
the stages
50
51
SADF apatite 6b);
these
pattern
c-axially
of mineral
electron
both
and d i r e c t l y
gap and o v e r l a p
field
images.
deposition b y SADF
order
within
t h e gap and
the pattern
to relate
of apatite
the stained
Certain
technical difficulties
because
i t i s known t h a t
demineralize adjacent
sections.
to mineral as regions
structure
distribution alcoholic
computer
deposition
with
this
was
patterns model
areas must
UA
fibrils
found
be
regions i n their
t o enhance t h e
were
PTA/UA
and a l i g n e d
to a
region
of a collagen
a different s t a i n s . So
of
charged
representative
fibril
fibril
with
o r i e n t a t i o n of
a t h e o r e t i c a l model
scaled
native
stained
s t a i n i n g produced
the molecular
of the stained
totally
and t o r e t a i n t h e sections
of
overcome
that
t o t h e aqueous
stain,
pattern
o n t h e same
a r e not as c l e a r
crystals,
determine
i t i s
banding
more d i s t a n t . T h e r e f o r e ,
as compared
non-mineralized banding
,field
s t a i n i n g procedures
i t was
T h e a l c o h o l i c UA
pattern
acids
Also,
of apatite
UA.
mineralized
aqueous
of collagen
unambiguously
amino
with
collagen
fibril.
collagen
the
orientation of
deposition
areas
to
that
overlap
a s by b r i g h t
the molecular
non-mineralized
banding
reverse
I t i s apparent
i s t h e same
to correlate
with
necessary
fine
in
(Figure
microscopy.
collagen
banding
visualizes
zones
oriented' c r y s t a l s appear
to the bright
demonstrated
In
identifies
crystals within
contrast
zones
specifically
(Figure
and t h e
are i n r e g i s t r a t i o n , thereby
7a).. The
scaled
enabling
the
52
determination
o f IM- t o C-
directed
left
with
a l c o h o l i c UA
PTA/UA bands The
from
stained within
d band
However,
pattern
also
toward
banding
pattern
of apatite
asymmetric
Figures
distribution
C-
portion
adjacent
fibrils
(Figure
i n these
determined
by t h e i r
spaces
(Figure 7b).
the
zones
mineralized
by a
i s consistent molecules
terminus.
was
t h e gap zone t h e
i s created
space
lucent with
also sides
orientations have
the
a n d when
I t was o b s e r v e d
molecular
on o p p o s i t e
PTA/UA.
7 b ) . The
f o r unstained
staining pattern
positioned
as w i t h
gap and o v e r l a p
of mineral
opposite
relative
of the f i b r i l s
7b a n d 5 a ) . W i t h i n
to the
having
period.
by t h e c h a r a c t e r i s t i c
o r i e n t a t i o n of the collagen i s nearest
pair of
of the D
their
stained
i s replaced
The p o s i t i o n i n g o f t h i s
apparent
lucent
maintained
t h e same a s w a s o b s e r v e d
(compare
to the
i t s characteristicintensity.
distribution
of apatite
observed
of a d i s t i n c t region
not as i n t e n s e l y
pattern
bands
s i m i l a r i n appearance
the mineralized
stained
precisely
and
and c o n s i s t e d
retained
Advancing
to
The
t h e a , e, a n d c b a n d s
distribution
o r i e n t a t i o n which i s
t h e more e l e c t r o n - d e n s e
b u t were
space.
to right.
s t a i n i n g were
positions
section
molecular
that as
consistent
o f t h e gap
zone
53
Figure
7. A n a l y s i s
turkey
tendon.
7a. A non-mineralized
fibril
stained
with
insert) acids
displaying
has been
molecular
the s p a t i a l
scaled
stained
opposite
banding
mineralized apatite border
opposite
with
shows
of the unstained t h e gap zone
t h e same p e r i o d i c sections.
and w i t h i n
(8a) and u n s t a i n e d
characteristic conditions sections
pattern
are stained
that
with
UA
reflections
are readily
reflection,
and t h e combined
reflection.
by
their
areas.
The
distribution
of
spaces
t h e gap zone
lucent
spaces
consistent
mineralized
i n 100%
stained
tendon
i s observed
apatite
with
X120,000.
o f a l c o h o l i c UA
of apatite
demonstrating
adjacent
lucent
asymmetrically
diffraction
7b.
Electron
o r i e n t a t i o n . Bars=100nm;
mineralized
The
i n the non-mineralized
the
8. E l e c t r o n
t o C-
i s not
as i n d i c a t e d
are positioned
Figure
t h e N-
amino
o f t h e same
a l c o h o l i c UA.
(arrowheads) op.posite
of charged
b and d bands.
portions
orientations
patterns
portion
image (7a
to determine
by t h e p r o m i n e n t
collagen
have
A computer
collagen
s t a i n i n g but the o r i e n t a t i o n i s
and m i n e r a l i z e d
fibrils
of a
staining pattern
Non-mineralized fibril
portion
distribution
and a l i g n e d
t o t h e PTA/UA
determined
orientation i n mineralized
a l c o h o l i c UA.
o r i e n t a t i o n . This
equivalent readily
of collagen
under
i s retained methanol.
i n d e n t i f i e d : the inner
(8b).
Two
The
both
when prominent
(002)
( 2 1 1 , 112, 300, 202, 301)
54
55
Electron mineralized
diffraction
areas
of
a l c o h o l i c UA
the
c h a r a c t e r i s t i c maxima
and
thereby After
confirming
influence the
best
was
the
reverse
collagen
fibrils
spatial
these
side
as:
of
amino
acids
the
contrast
that
densities as
white
of
hydrophobic the
positioned
gap in
terminus
of
collagen
fibril
hydrophobic intermediate
zone;
the
the
9a).
gap
are
in
zone
molecular containing
amino
acids
regions
direction
a^-a^
third
and
lies
between
groups. of
in
model
include
l i e within
from
with
the
which
c^-c^,
and
high
contrast
and
spatial
the
fibril
are
image.
border
Two
each
asymmetrically
a^
most
having
normal
e^
nearer
o r i e n t a t i o n . Regions low
These
their
collagen
acids
reverse
amino
negative),
the
regions
amino
of
by
various
observed
three
the
regions, the
these,
I t was
b).
analyzed
because
displayed
hydrophobic
areas
Of
crystals
the
hydrophobic
mineralized
acids
there
further
selected
unstained
(Figure
of
showed
and
( p o s i t i v e and/or
structure.
amino
nature
8a
selecting for
and
were
C-
was
models
charged
collagen
model
to the
aromatic,
hydrophobic
displayed of
such
on
N-
distribution
computer
f i t to
computer
apatite
molecular
to
of
biological
dense
sections
the
non-charged,
classes
stained
determining
groups
polar
these
(Figure
apatite-collagen
acid
for
from
i t s presence
mineralized
comparison
analysis
of
of
densities the
overlap
gap zone
the the
of zone; from
c
1
to
56
a^,
and
within
the
gap
computer-generated with 9b
micrographs
and
that
inset).
apatite
spatial
the
gap
overlap and
D
gap
(1.93) the
zone
percentage
of
are: the
1*1 = 1 1 % ,
organic (see
composition
by
high
composition
of
the
collagen
hydrophobic, repeating
there are
of
129
and
100
as
o v e r l a p zone
amino
4%.
energy
of
the
the
differences
these
two
zones
140)
overall
relate
the
amino
varies zone
the
V=27%, percentages
figures
water
of
to
for an
acid hydrophobicity
differences
i n amino about
acid 0.1%.
hydrophobicity throughout
differences
in composition.
Using from
ratios
gap
the
in
twice
acids
h y d r o p h o b i c i t y by
i n average to
1= 14%.
transfer of
The
a r e : 1*1 = 9%,
o v e r l a p zone
acids
about
acids. amino
acids
amino
W i t h i n the
acids
F = 16%,
p.
amino
the
i n width
amino
has
of
shows
than
5 molecules
hydrophobic
most
a measure
1980,
average,
hydrophobic
at
D period
hydrophobic
hydrophobic
by
on
unit,
L = 34%,
Thus,
differences
occupied
acid
free
vary
areas
shown
amino
V = 25%,
Tanford,
i t i s clearly
hydrophobic
zones
solvent
(Figure
the
1=17%. W i t h i n t h e
additional
collagen
residues.
hydrophobic
L=30%, F=17%,
correlate
acid
constituent
two
images
and
amino
density
the
These
repeats align
i n those
T h e r e f o r e , the
individual
c^*
hydrophobic
I n one
zone.
to
of
o v e r l a p zone
the
between
dense
i s less
zone.
the
set of
o v e r l a p zones
in length,
within the
and
e^
unstained mineralized
this
densities
gap
that
of
In
of
from
hydrophobic
i s less
Analysis
zone
in density
and
not
to
57
The the
proportions
D u n i t were
each
zone;
zone.
This
101
o f gap and o v e r l a p
c a l c u l a t e d by c o u n t i n g f o r the overlap
results
i n the predicted
occupied
by t h e o v e r l a p
be
assuming
.568D
are
equivalent.
the
density
for
length
that
These
o f amino of each
triple
residues
acids
density
of these
the
gap zone
gap
zone
have
regions
.432D
acids for
of the D
unit
and t h e gap zone
occupied then
1 0 ) . The
by a m i n o
be u s e d and t o
density
of s t a b i l i t y
zone
there
residues
(1.13:1).
may
proportion
to
acids
to predict
standardize
compared
regions,
localized these
molecular
and p r o l i n e t o 335 w i t h i n t h e
i s a greater
i n the overlap
Two
of
of the
a r e 359 h y d r o x y p r o l i n e
Therefore,
very
to
and 133 f o r t h e gap
per molecule
within the overlap zone.
unit.
figures could
(Figure
There
overlap
These
the lengths
are a reflection
helix.
t o be
t h e amino
and p r o l i n e a r e p e r i o d i c a l l y
the D period
residues
zone
relative
zone.
Hydroxyproline along
zone
zones
proportional
zone
as compared
one a t e a c h
few p r o l i n e o r h y d r o x y p r o l i n e represent
more
flexible
to
end o f t h e residues.
portions
of the D
58
Figure amino
9.
A comparison
acids
and m i n e r a l
hydrophobic reverse left
amino
acid
high
as
white.
as b l a c k
shown
with
image
of hydrophobic
an a l i g n e d
amino
mineralized
and s c a l e d amino
reverse
acids
hydrophobic mineral
mineral amino
density,
hydrophobicity. Figure
structure
have
corresponds
acids; while correspond
Bar=1QQnm;
These
amino
of collagen
lucent
The
acids
may
residues
accomodate
computer
asymmetrical
with
low i n less
high i n
confer This
Regions
in
image
with
to the
indicates that
residues
of collagen
are considered
to changes
hydroxyproline
stability
to these
i m p l i c a t i o n s on t h e a d a p t a t i o n
hydroxyproline
areas,
i s
registration
The
o f p r o l i n e and
molecules.
growth.
fibril
image
X12Q,0Q0.
i s a periodic localization
apatite crystal
contrast
to regions
to regions
10. A computer image
distribution.
there
(density)
depicted
contrast
(inset).
i s from
fibril
acids
collagen
d e n s i t i e s o f t h e two i m a g e s c o r r e s p o n d .
localized
and
of the c o l l a g e n
of
and
orientation
and i n t h e r e v e r s e
9 b . An u n s t a i n e d
hydrophobic
i n normal
molecular
d e n s i t i e s of hydrophobic
t h e t o p image
to
t o C-
of
9 a . A computer image
groups displayed
Periodic regions
in
and
apatite.
contrast; the
to right.
contain
of the d i s t r i b u t i o n
fewer
may
molecules
p r o l i n e and
t o be m o r e
conformation.
which
flexible
59
60
DISCUSSION
The
ordered
collagen
has
distribution
been
the
interactive
the
biomechanical
distribution collagen how
limited.
of
has
of
long
date,
focussed
may
potential
within
the
gap
from
the
overlap
mineralization comes
from
(Hodge
apatite zones
during
process.
electron
diffraction
s t u d i e s (White
specific
and
imaging
has
overlap
zones
mineralization recent
findings
localization sequence
of
direct
shown at
to
1966), et
to
earliest
a
the
present
the
molecular
collagen;
and
of
This nature
of
knowledge
of
collagen i s
result
very
of
P e t r u s k a , 1963;
Glimcher,
b e l i e v e d to early
stages
support
neutron
be
excluded
of
the
this
viewpoint
1959),
electron
diffraction
a l . , 1977).
and
A recent apatite
by
present
i n both
gap
As
an
orientation
consequently
on
SADF and
of
e x t e n s i o n of
compares
x-ray
study
of
detectable stages
study
be D-stagger
(Glimcher,
be
to the
to
( A r s e n a u l t , 1988). the
our
I
producing
the
presumed
visualization
crystals the
with
been
microscopic
(Engstrom,
the
as
Evidence
diffraction
time
space
and
has
because
tissues.
reflect
this
i n type
mineral/collagen relationships
the
zone
study
collagen in
to
interact
t h e o r i e s of
crystals
skeletal
at
on
Therefore,
and
viewed
However,
collagen molecules
1983).
mineral
been
apatite
s u b j e c t of
p r o p e r t i e s of
crystals
To
available
important
role
structure.
apatite
have
an
of
these
apatite and
offers
amino a
acid
hypothesis
61
of
collagen
both
bright
early
influence
on
field
selected-area
stages
of
characterized apatite the
of
by
had
gap
zone
of
either
side
fibril.
of
and
midportion
properties
of
Regions
gap
the of
confinement
to
involved
the
result has
a
of
in
of
these
of
apatite
D
in
period
which
position
acids
as
which
apatite gap
to
depicted have
the
regions
evidence
of for
crystals in zones.
mechanism
of
Thus,
exclusion
from
implications
on
the
gap
zone the
crystals within
the
periodic
both
zones.
least
mineral density
computer
less
of
modelling.
mineralization
hydrophobicity.
a more collagen
complicated than
hydrophobicity
these
on
the
greatest
collagen
most
d i r e c t i o n of
periodic by
of
that
have
the
along
zone.
observations
within
provide
the
terminal
of
fibril.
regions
within
fibril
the
apatite
number
from
p o s i t i o n to
of
the
regions
consisted
discrete area
that
distribution
overlap
collagen
regions
distribution
an
the
reflects
Conversely,
findings
of
by
are
collagen
mineral
were
and
the
likely
amino
These
of
imaging
specific
most
hydrophobic
in
zone nearer
zones
correlate
correlate
density
distribution
overlap
density
and
concluded
heterogeneous
to
observed
field
periodic
o r i e n t a t i o n of density
positioned
I t was
and
greater
the
dark
corresponds
the
I t was
mineralization
asymmetric
least mineral
invariably
gap
an
molecular
which
the
Areas
C-
formation.
intrafibrillar
c r y s t a l s which to
Areas
and
bone
simple may
mineralization domains.
This
the i n t e r r e l a t i o n s h i p
be as
a
work
62
between
the
mechanism
architecture molecular
of
are
high
and
correlate
or
in
prevent
density
be
determined
hydrophobic
groups. likely
Local to
apatite.
be
molecules
in
various the
the
Various
1985b),y and
associated
with
the
amino
of
site
such
or
macrornolecules
et
either
nucleation
of
and
the
phosphate
hydrophobic
these of
may
and
calcium
of
ions
are
biological
fibril
may
be
non-collagenous
acid
as
on
(Scott
association
such
carboxyglutamic
less
influence
proteoglycans of
mineral
with
positive control
sites
(Hauschka
formation
are
diffusion
collagen of
a^-e^)
i s o l a t e d from
nucleation
as
We
of
association
the
and
regions
acids.
gradients the
c^-c^
e f f e c t may
the
fibril
Conversly,
in
including
negative
specific
osteocalcin
the
by
crystal
area.
occur
localization
have
and
collagen
(a^-a^>
environment
close
for
the
initial
ions
properties
have
the
example,
Macromolecules,
collagen.
1979)
For
by
defined
inhibit
to
the
specific
critical
which
1985)
into
concentration
Also,
instrumental
growth.
a
affected
may
hydrophobic
First,
by
of
which
expected
the
as
along
acids
hydrophobic
areas.
localization
(Veis,
of
processes.
amino
growth
be
that
two
be
localized
areas
can
of
Haigh,
to
mineralization
collagen.
regions
crystal
speculating
will
of
collagen
distribution
hydrophobic
distribution spatial
crystal
ordering
Specific
of
crystal and
with
phosphoproteins
(Glimcher
a l . , 1975)
collagen-rich mineralizing
et a l . ,
have
tissues.
been
63
Secondly, by
space
apatite crystal and
energy
hydrophobic require
a
mineral.
groups net
The
these or
two
free
during
result
being
regions
state achieved groups
and
i s mutually
neither
potential
with
processes
Although
there
hydrophobic
not
does
some D
periods
visualized
within
the
zone.
gap
In
which
hypothesis. the
of
other
Several computer
regions.
Rather
class
amino
acids
must
of
to
crystal
are
not
result
of
the
medium
also
one
6).
(Figure
9)
of
show
i s depicted
linear more as
1980);
mineral
along
a^-e^
these
line
"Why
in
is
the
the and
the
mineral
proposed are
possible.
that
homogeneous w i t h i n the perfectly
of
In a d d i t i o n ,
examine
explanations
favorable
these
defined
this
of
influences.
question
to
Both
(Tanford,
the
more
must
the
points
exibit
may
interaction
exclusion
the
of
conditions
addition,
(Figure
ask
growth.
concurrent
be
will
hydrophobicity
minimizing
exceptions
images
influenced
distribution
apatite in collagen
potential
a
the
of
a
exclude
words,
i s not than
as
aqueous
do
be
environmental
s e c t i o n s not
represent
composition
of
One
mineralized
consistent?"
acid
areas
i s present
distribution
unstained,
by
there
where
First,
affect
local
seem
regions
may
dissociation
e x c l u s i v e . In
do
mineral
The
optimal
the
regions
crystals
that
whether
hydrophobic
periodic
will
of
growth
m i n e r a l i z a t i o n process
c o n s i d e r a t i o n s , occur
energy
from
the
which
processes,
energy
and
requirements.
energy
compartmentalize
size
the
amino
hydrophobic
arrangement, clumps.
This
this
64
concept
i s compatible
hydrophobicity aggregate.
with
i n that
Therefore,
thermodynamic
the hydrophobic i t may
regions
amino
a c i d s . There
acids
( i na three-dimensional crystal
regions.
may
formation
Second,
amino
i n the density
be s u f f i c i e n t
tend
of
hydrophobic
arrangement)
of these
the three-dimensional
packing
arrangement
and d i s c o n t i n u o u s
et
a l . , 1985).
(1963) model
to hold
collagen
molecules
approximately a
70nm
thick
forming means
true
70nm
f o r the relationship
visualized thick.
s e c t i o n may
outside
be o f f s e t
of the general
thicker
the section,
regions
w i t h i n t h e gap zone
because
the space
regions) thickness space tilted
of
crystals
of the section.
a t 0°would the space
greater. longer
between
( i nthis
pass would
At a c e r t a i n
permit
regions
to result
molecules
distortion.
there
Third, the
relative
This i s
hydrophobic to the
electrons entering the
but i f the specimen smaller
were
as t h e angle
of incidence
the space
became would
o f e l e c t r o n s . The p r e d i c t e d based
i s no
the electron lucent
the proposed
Incident
of
i n crystals
n o t be v i s u a l i z e d .
are small
angle
the passage
the hydrophobic
case
appear
n o t be
between a l l
sufficient
likely
will
through
could
p a t t e r n . However,
of this
t h e more
(Hulmes
i n a s e c t i o n which i s
Therefore,
to p r e d i c t the degree
permit
hydrophobic
c o l l a g e n i s somewhat d i s t o r t e d
expected
amino
to
of
The H o d g e - P e t r u s k a
to
to describe
non-hydrophobic
packing
w i t h i n each
of
acids
be more a c c u r a t e
these
some
as d i f f e r i n g
concepts
on t h e c o m p u t e r
no
size
models
65
(a^-a2=.12D a^-e2=.07D
o r about or about
7.8nm;
(70nm) g i v e s
the
would
mineral
within
specimen region
incident spaces, angle
a critical
no l o n g e r
positioned
t h e gap zone
angle
would
region,
o f 7.6° T h e r e f o r e , t h e gap zone
would
would
than
sectioning
becomes c r i t i c a l l y
this
the region
would
because
o f i t s more
collagen
fibrils.
Electron represents component The
microscopic
a technical while
approach
aqueous
PTA/UA
banding
pattern.
the with
bands
imaging
visualizing
groups,
more
as does
The a n g l e o f the normal
the tendon.
t o image
the associated
with
UA i n a l c o h o l
And s o
i n bone of
tissues
the mineral organic
matrix.
i s an effects of
a different
collagen
t h e p o s i t i o n i n g and r e g i s t r a t i o n of
" a t o e" a r e t h e same
charged
much
to maintain
a t an
regions
of calcified
but produces
While
predicted
be v i s i b l e
to the demineralizing
stains
electron
structural organization
challenge
staining of collagen
alternative
within
be m o r e d i f f i c u l t complex
t h e a^-e^
t h e gap zone.
important
that the
tothe
a t an
of these
no l o n g e r
within
i n direction of collagen
pattern
equal
disappear
be d e t e c t e d
a t which
Assuming
the non-mineralized
frequently
change
angle
i n parallel,
o f 3.5? The l a r g e s t
t h e c^-c^
bordering
are blocks,
9.4nm; a n d
specimen
incident
be d e t e c t a b l e .
t h e gap zone
i n thickness,
within
o r about
4.7nm) a n d a n e s t i m a t e d
thickness space
0^-0^=.14D
d u e t o t h e i n t e r a c t i o n o f UA
the relative
i n t e n s i t i e s and
66
delineations of
the
of
observed
reasons.
First,
UA
a
to
and
the
be
bands
pattern
i s not
although
charged and
Secondly,
e f f e c t s of
the of
comparison
The collagen
each
between has
not
would
alcohol
as
a
been
water
and
alcohol
and help
scales,
as
of
the to
degree
despite
thus
overlap
zones
evidence
which
crystals
within
the
for
r e l a t i o n s h i p because
periodic
and
a l . , 1982b). in
the
so
a in
there
collagen
spaces
patterns
this
the
in
precise between within
proposed
availability
of
has
assigned
not
been
calculations in
is in
degree
contradicts gap
et
hydrophobicity
collagen
molecules.
estimating is a
the
several a
the The strength
subjective
of f i t .
localization of
of
making
v i s u a l methods
the
consider
on
correlation
substantiate
impossible
of
two
negatively
solvents
more
A mathematical
hydroxyproline
scales,
both
solvent
studied
for
done.
preliminary
However,
interpretation
the
(Tzaphilidou
not
is a
explanation
staining
depending
has
been
mineral
method
The
acids
variables
methods.
these
limitation the
UA
to
hydrophobic
of
of
fibrils
relationship.
standard
works
repeating
of
on
c l e a r . An
v i s u a l i n t e r p r e t a t i o n of
region
value
pH
collagen
correlational density
as
r e a d i l y apparent
early
amino
concentration
procedure
not
cationic stain i t actually stains
positively
staining
are
of
apatite
fibrils
theories (eg.
crystals
(Arsenault,
exclusively
UJeiner
and
within 1988)
is
localizing
Traub,
1 986)
or
67
spaces
within microfibrils
comes f r o m variable
studies
and
not
(Arsenault
and
spaces
be
may
localization explain given
the
mineral
of
crystal
These
molecules
chain
flexibility
helix
can
have
its
a
accommodate
in free
and
microscopic
stability)
2)
p r o l i n e and
4)
charged
are
et
growth
form
unit.
So
densely
or
gives
how
the
form
a
et
on:
a l . , 1981). fibrils
the
apatit zone
semiflexibl
and
1985a). side
the
triple
These which
according
studies of
G-X-Y
localization;
chain
the
appreciable
flexibility 1)
crystal
(Veis,
because
biomechanical
of
order
backbone
without
be
to
overlap
as
molecule
flexibility
The
rise
packed
higher
of
not
does
conceptualized
collagen
lateral
side
space
potential
heterogeneity
a l . , 1987)
dependent
acid
and D
hydroxyproline
amino
these
which
degree
G i r a u d - G u i l l e , 1988).
gap
therefore
(Okuyama
and
the
must
fluctuations
i n t e r t w i n e to
of
is
Consideration
collagen
high
energy
size
size
do
be
the
(Sarkar
longitudinal
electron 1980;
have
crystal
evidence
they
i n t e r t w i n e to
cylinder,
that
Although
i n the
can
Other
materials
entire
peptides
1980).
in crystal
size
deposited
which
molecules
1988).
d i s c r e t e environment
Collagen
changes
the
non-collagenous
w i t h i n the
semiflexible
shown by
observations.
become
cylinders
determined
Grynpas,
of
heterogeneity deposition
have
influential
these
to
that
(Hohling,
also to
(l/iidik, collagen
(an
triplet; 3)
interactions;
crosslinking and
68
5)
hydrophobic
collagen (Veis,
molecules
by
modelling flexible
i t can
overlap
Some o f
the
overlap
zones
chain
flexible
be
built
observed that
the
of
low
into
repeating
gap
zone
gap
d e n s i t i e s of regions
density
of
hydrophobic
regions
of
high
zones
mineral
by
the fibrils.
i n gap
acids
and
localized
by
regions
hydroxyproline
to
and
regions are
discussed
that
is
density
i s bounded
correspond
hydrophobicity
flexible
differences in
zone
and
this
w i t h i n the
p r o l i n e and
amino
units
periodic
mineral
molecules
by
are
the
regions
From
i s more
overlap
explained The
10).
there
collagen
Thus,
inflexible
that
the
collagen
be
c ^ - c ^ ) . These
and
(Figure
d i f f e r e n c e between may
interactions.
It i s possible that
flexibility.
very
side
modelling
and
into
semiflexibility
and
be
zone.
accommodated
with
can
computer
regions
collagen
acid
exibit
1985a) which
depicted
the
amino
with
adjacent
(a^-a^ a
low
to
the
above.
Conclusions
In it
would
an
observational, correlational
be
relationship Based this
on
exists
previous
t h e s i s are
experimental clarify in
improper
the
to
suggest
between
studies
the
the
biologically
designs potential
m i n e r a l i z a t i o n . In
are
that
of
cause
such and
v a r i a b l e s under
as.this,
effect examination.
relationships described p l a u s i b l e but
required
role
a
study
to
conclusion,
the
alternative
substantiate
hydrophobicity
in
and
asymmetric
and flexibility and
69 periodic and
d i s t r i b u t i o n s of
overlap
zones
correspond' to distribution
of
collagen
collagen has
a
apatite
fibrils
molecular
reverse
crystals within were
the
observed
o r i e n t a t i o n . This
c o r r e l a t i o n to
the
to mineral
position
hydrophobic
groups
as
determined
by
computer
modelling
may
intrinsic
properties
of
collagen
structure
reflect
throughout
the
found
periodic
that
flexibility
gap
may
apatite
into
role
collagen
be
given
to
throughout potential
of
the
the
collagen
mechanisms
mineralized
the
of
tissues.
addition,
i t
of
proline
Therefore,
properties rather
the
gap
of
than zone.
may
and
was
and
to
collagen
Further of
b i o m i n e r a l i z a t i o n and
as
on
the should
they
vary
the
study
apatite
enhance
of
understand
focussing
localization fibrils
of
intrafibrillar
mineralization, consideration
discrete
within
in
In
p r e f e r e n t i a l accommodation
regions.
period
zones.
density
explain
between of
by
during
D
space
properties
differences
specific
the
association
overlap
determined
hydroxyproline,
of
and
gap
of
the
and
our
understanding
the
mechanics
of
70
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amino
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Methods
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