BIOCHEMICAL
Vol. 79, No. 2, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OXYGENPARTICIPATION IN THE IN VIVO AND IN VITRO AGING OF COLLAGEN FIBRES B.J. Rigby, T.W. Mitchell
and M.S. Robinson.
Division
of Textile Physics CSIRO Ryde N.S.W. 2112 Australia Received
September
26,1977
Summary Wehave examined, as a function of oxygen concentration, the in vivo and in vitro aging rates of tendon from animal species with widely differing life-spans. The results suggest that concomitant with the genetically determined aldimine type cross-linking reactions which probably are complete by the time the animal has reached physiological maturity, there is an on-going oxygen-mediated system of reactions which also effectively cross-link the structure. These reactions appear to proceed with their own intrinsic rate dependent only upon oxygen concentration, and independent of the particular species involved. They may not be required by connective tissues, but are an unavoidable consequence of the presence of free oxygen in tissues with a low rate of turnover. Introduction
mechanical and chemical changes which take
The physical,
place in collagenous tissues with in vivo aging are well known (1).
It is now well established
graphic example is the decrease in solubility. that similar reconstituted consolidation;
changes take place in vitro (4) collagen fibres.
for both native
These aging effects
the tissue takes on the characteristics
has becomeprogressively
bound by intermolecular
is known about someaspects of the cross-link for exmaple references 5 and 6), generally
or hydroxylysyl
residues.
condense with reactive
are processes of of a system which
cross-links.
While much
chemistry of collagen (see
the story is far from complete.
of cross-links
initially
This is the oxidative
The resulting
It is
deamination of lysyl
aldehydes are then believed to
groups such as the c-amino group of hydroxylysine
in neighbouring molecules.
However, it is not clear whether the number
laid down remains constant,
and their
changes with time, or whether the number of cross-links
0 1977
(2,3) and
agreed that only one enzymatic process is necessary to begin
the formation of cross-links.
Copyright AN rights
A
by Academic Press, Inc. in any form reserved.
of reproduction
reactivity
increases with
400 ISSN
0006-291
X
BIOCHEMICAL
Vol. 79, No. 2, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TEMPERATURE
Fig.1. Isometric melting curves from an animal killed at 40 days. A. B.
No in vitro 180 days 60 n mol 180 days 300 n mol
C.
time.
Apparently
have
shown that
and human tissues A further
unanswered
are the
same process.
tendon
rate
collagen
of aging
amount
for
with
age the
could
collagens,
in the incubating
solution
Bailey
process
in 0.9% Nacl
containing
and Shimokomaki
is
complete
that
in vivo.
increased,
by about
the
concentration
compared
the
amount
tissue
fluid.
Also,
the rate
to the
in vivo
rate
by adjusting
of aging the
in vitro free
401
oxygen
aging
human and
same
of free
be brought
concentration
the
the
available
could
bovine
Further,
by increasing with
(7)
by maturity.
and in vitro
evidence
same rate
be greatly
containing
in young rat,
in vivo
(3) we presented
age at much the
both
tendon
cross-links
is whether
paper
in vitro
tail
terminate; derived
question
In a recent rat
aldehyde
change
rat
aging in vitro aging in 0.9% Nacl ml-l oxygen, 20°C. in vitro aging in 0.9% Nacl ml-l oxygen, 20°C.
some processes while
for
-C
oxygen
in the close to body tissue
Vol. 79, No. 2, 1977
level.
These
melting
curves
are
effects
in vivo -1
300 n mol ml
would
-1
be obtained
three
plays
a significant
Curve
curve
(tissue
with
which
in the
similar
the
tendon
with
C was incubated
to the
curve
which
at 40 + 180 days
based
upon mechanical
supports
aging
for
incubated
curve
killed
evidence,
collagens, role
while
C is
at two different
curve
the tendon
level),
from a rat
further
different
for
isometric
aged 40 days at death
the melting
Curve
tendon
where
180 days in saline
A is
fluid
in Fig.1
an animal
B that
of oxygen.
We now report using
from
at 20°C for 1
aging,
oxygen
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
are summarised
tendon
of oxygen.
60 n mol ml with
tail
incubation
concentrations only
of oxygen
of rat
shown after
with
BIOCHEMICAL
the view
process,
both
experiments
that
in vivo
(3).
free
oxygen
and vitro.
Methods The extent determined
of aging
on collagen
were
extended
they
broke.
fibres
in an Instron
The collagen and human wrist
to yield
animals
widely
approximately. so the
fibres
were made:
from
of extension,
curves
Figs.2
with
similar
The fibres -1
until
and dog tail
amino-acid but
from
3, 15 and 80 years a day or two after
effects.
and.from
the
linear
At least
curves
part
five
two measurements
of the
curve,
and
breakage.
and 3 give against
of rat
systems)
within aging
at 36’C.
tendons
namely
of the
before
plotted
from
curves
of 1.2% min
cross-linking
tested
modulus
and in Fig.3
taken
to in vivo
attained
at a rate
life-spans, were
load/extension
saline
collagens
each sample
and Discussion
load/extension
1.
refer
the extension
the maximum stress Results
differing
results tested
were
similar
The samples
death
were
Extensometer
mammalian
(and presumably
by simple
in physiological
fibres
composition with
was gauged
data
animal
the maximum stress
derived
age;
from the
in Fig.2
developed
the modulus
before
This data in non-graphical form, is referred to in Ref.3 of the experimental procedure are given there.
402
the
fibre
and details
BIOCHEMICAL
Vol. 79, No. 2, 1977
AGE
02
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
03
IN YEARS
0
10
20
30 LO AGE IN YEARS
50
60
The modulus of the load/extension curve as a function of Fig.2. 1 and dog tail A, age for tendon from rat tail l , human wrist extended in 0.9% Nacl at 36OC, at a rate of 1.2% min-1 until breakage occurred. Fig.3. The stress at break attained in the load/extension as a function of age, for the same samples and conditions in the legend to Fig.2.
broke.
It
connects if
is apparent
values
the rat follow
three
collagens
the
in a manner
taken
from
earlier
under
the
process were
appear
in vitro
in very the young
for
stress
in a curve
three
the aging
(as far
killed
parameters
for the
increased
at break),
would well
for
oxygen
increase
within
with
x 10’ while
free
limiting
experiments
upon tail
dynes the
older
403
in Ref.3. available
values
rat
tendon
and whose tendon According
to
to the tendon rate
so that
The results
shown
modulus
are concerned) As a check
2.5 years.
cm -2 for
the
life-span.
the aging
the
words
properties
with
as described
seems that collagen
In other
load/extension
2.5 years
condition
(5.6
of their
as mechanical
smoothly
and it
at the age 50 days and 23 months
be completed
rat:
which
species,
human collagen.
we performed
good agreement
for
result
of the
as for
to age
in vitro
findings
would
plots
seems to have no connection
two rats
aged
longer
same curve
which
both
the collagen
interpretation
had been our
from
and dog lived
would
of this
that
curve listed
in Figs.2
the obtained and 3
and 3.6 x lo8 dynes
the modulus
agreement
is
cm -2
70
Vol. 79, No. 2, 1977
excellent (2.4
(4.8
BIOCHEMICAL
x lo’),
and the
of 54 year whereas
old
(3)
aging,
is work
more,
showed
authors
conversion
because
decrease
All properties
the
is reasonable
growth,
oxygen-dependent
background
of free
Finally, of connective the problem
maturity,
raised
be “toughened”-in
biological fact
most
could
forms
or without
oxygen.L changes
if
some of the (6,7)
throughout
are
is
reaction. the
aging
This
is
genetically by
In any case,
life. should
considered
a
of
are completed
tissues
It should be pointed out here that insolubility, and the magnitude of the stress and temperature of the fibre in an isometric melting experiment, co-valent cross-linking state of the fibre i.e. if the number and/or the thermal and mechanical cross-links increases.
404
with
at least
why the
effects
associated
with
constant
a cross-linking
has relevance.
all
concerned
the nature
work
not
categories
from the
Whatever with
in the
The other
solely
problem
that
system
of tissues.
consistent
reason
not be due to
age-related
fluid.
continues
aging
with
resulting
4 months
Further-
to more stable
determined
the present
process
after
be due to two distinct
tissue
reactions,
tendon
of insolubilisation
time
the
aging,
a marked
with
and repair
evidence
showed
upon oxygen.
that
a general
in vitro
dependent
place
genetically
is
which
by the
the aging
no obvious
is
for
cross-linking yet
is
there
tissue
took
could
in the
end result
rate
cross-links
mechanism
oxygen
its
also
suggest
maturation
continuous
the
changes
bonds
the
human tendon
of this
is
tissues
One is
mechanism,
old
curve
by 7 months
hypothesis.
the
in these
altered
melting
support
aldimine
of collagenous
formation,
determined
that
isometric
of “old”
above results
of cross-linking.
2.
at break
characteristics
fibres
of reducible
the
was not
(8) has shown that
collagen
there
stress
the
5.5 year
further
of reprecipitated the
for
the melting
Recent
this
for
that
tendon
curve
toward
of in vitro
finding
human wrist
the melting
alteration
the
agreement
x log). Our earlier
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
continue
to
to be undesirable.
mechanical strength attained before breakdown are all indices of the these parameters increase stability of the
Vol. 79, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The unavoidable oxygen-mediated reactions which we have postualted, provide an explanation for this problem.
References 1. Sinex, F.M. (1968) in "Treatise on Collagen", Gould B.S.(ed.) ~01.2, part B, pp.409-448, Academic Press, New York. 2. Rigby, B.J. (1967) Biochim. Biophys. Acta 140, 548-551. 3. Mitchell, T.W., and Rigby, B.J. (1975) Biochim. Biophys. Acta 393, 531-541. 4. Gross: J. (1963) Biochim, Biophys. Acta 71, 250-252. 5. Tanzer, M.L. (1973) Science 180, 561-566. 6. Bailey, A.J., Robins, S.P., and Balian, G. (1974) Nature 251, 105-109. 7. Bailey, A.J., and Shimokomaki, M.S. (1971) FEBSLetters, 16, 86-88. 8. Robins, S.P., and Bailey, A.J. (1977) Biochim. Biophys. Acta 492, 408-414.
405
would
Vol. 79, No. 2, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OXYGENPARTICIPATION IN THE IN VIVO AND IN VITRO AGING OF COLLAGEN FIBRES B.J. Rigby, T.W. Mitchell
and M.S. Robinson.
Division
of Textile Physics CSIRO Ryde N.S.W. 2112 Australia Received
September
26,1977
Summary Wehave examined, as a function of oxygen concentration, the in vivo and in vitro aging rates of tendon from animal species with widely differing life-spans. The results suggest that concomitant with the genetically determined aldimine type cross-linking reactions which probably are complete by the time the animal has reached physiological maturity, there is an on-going oxygen-mediated system of reactions which also effectively cross-link the structure. These reactions appear to proceed with their own intrinsic rate dependent only upon oxygen concentration, and independent of the particular species involved. They may not be required by connective tissues, but are an unavoidable consequence of the presence of free oxygen in tissues with a low rate of turnover. Introduction
mechanical and chemical changes which take
The physical,
place in collagenous tissues with in vivo aging are well known (1).
It is now well established
graphic example is the decrease in solubility. that similar reconstituted consolidation;
changes take place in vitro (4) collagen fibres.
for both native
These aging effects
the tissue takes on the characteristics
has becomeprogressively
bound by intermolecular
is known about someaspects of the cross-link for exmaple references 5 and 6), generally
or hydroxylysyl
residues.
condense with reactive
are processes of of a system which
cross-links.
While much
chemistry of collagen (see
the story is far from complete.
of cross-links
initially
This is the oxidative
The resulting
It is
deamination of lysyl
aldehydes are then believed to
groups such as the c-amino group of hydroxylysine
in neighbouring molecules.
However, it is not clear whether the number
laid down remains constant,
and their
changes with time, or whether the number of cross-links
0 1977
(2,3) and
agreed that only one enzymatic process is necessary to begin
the formation of cross-links.
Copyright AN rights
A
by Academic Press, Inc. in any form reserved.
of reproduction
reactivity
increases with
400 ISSN
0006-291
X
BIOCHEMICAL
Vol. 79, No. 2, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TEMPERATURE
Fig.1. Isometric melting curves from an animal killed at 40 days. A. B.
No in vitro 180 days 60 n mol 180 days 300 n mol
C.
time.
Apparently
have
shown that
and human tissues A further
unanswered
are the
same process.
tendon
rate
collagen
of aging
amount
for
with
age the
could
collagens,
in the incubating
solution
Bailey
process
in 0.9% Nacl
containing
and Shimokomaki
is
complete
that
in vivo.
increased,
by about
the
concentration
compared
the
amount
tissue
fluid.
Also,
the rate
to the
in vivo
rate
by adjusting
of aging the
in vitro free
401
oxygen
aging
human and
same
of free
be brought
concentration
the
the
available
could
bovine
Further,
by increasing with
(7)
by maturity.
and in vitro
evidence
same rate
be greatly
containing
in young rat,
in vivo
(3) we presented
age at much the
both
tendon
cross-links
is whether
paper
in vitro
tail
terminate; derived
question
In a recent rat
aldehyde
change
rat
aging in vitro aging in 0.9% Nacl ml-l oxygen, 20°C. in vitro aging in 0.9% Nacl ml-l oxygen, 20°C.
some processes while
for
-C
oxygen
in the close to body tissue
Vol. 79, No. 2, 1977
level.
These
melting
curves
are
effects
in vivo -1
300 n mol ml
would
-1
be obtained
three
plays
a significant
Curve
curve
(tissue
with
which
in the
similar
the
tendon
with
C was incubated
to the
curve
which
at 40 + 180 days
based
upon mechanical
supports
aging
for
incubated
curve
killed
evidence,
collagens, role
while
C is
at two different
curve
the tendon
level),
from a rat
further
different
for
isometric
aged 40 days at death
the melting
Curve
tendon
where
180 days in saline
A is
fluid
in Fig.1
an animal
B that
of oxygen.
We now report using
from
at 20°C for 1
aging,
oxygen
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
are summarised
tendon
of oxygen.
60 n mol ml with
tail
incubation
concentrations only
of oxygen
of rat
shown after
with
BIOCHEMICAL
the view
process,
both
experiments
that
in vivo
(3).
free
oxygen
and vitro.
Methods The extent determined
of aging
on collagen
were
extended
they
broke.
fibres
in an Instron
The collagen and human wrist
to yield
animals
widely
approximately. so the
fibres
were made:
from
of extension,
curves
Figs.2
with
similar
The fibres -1
until
and dog tail
amino-acid but
from
3, 15 and 80 years a day or two after
effects.
and.from
the
linear
At least
curves
part
five
two measurements
of the
curve,
and
breakage.
and 3 give against
of rat
systems)
within aging
at 36’C.
tendons
namely
of the
before
plotted
from
curves
of 1.2% min
cross-linking
tested
modulus
and in Fig.3
taken
to in vivo
attained
at a rate
life-spans, were
load/extension
saline
collagens
each sample
and Discussion
load/extension
1.
refer
the extension
the maximum stress Results
differing
results tested
were
similar
The samples
death
were
Extensometer
mammalian
(and presumably
by simple
in physiological
fibres
composition with
was gauged
data
animal
the maximum stress
derived
age;
from the
in Fig.2
developed
the modulus
before
This data in non-graphical form, is referred to in Ref.3 of the experimental procedure are given there.
402
the
fibre
and details
BIOCHEMICAL
Vol. 79, No. 2, 1977
AGE
02
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
03
IN YEARS
0
10
20
30 LO AGE IN YEARS
50
60
The modulus of the load/extension curve as a function of Fig.2. 1 and dog tail A, age for tendon from rat tail l , human wrist extended in 0.9% Nacl at 36OC, at a rate of 1.2% min-1 until breakage occurred. Fig.3. The stress at break attained in the load/extension as a function of age, for the same samples and conditions in the legend to Fig.2.
broke.
It
connects if
is apparent
values
the rat follow
three
collagens
the
in a manner
taken
from
earlier
under
the
process were
appear
in vitro
in very the young
for
stress
in a curve
three
the aging
(as far
killed
parameters
for the
increased
at break),
would well
for
oxygen
increase
within
with
x 10’ while
free
limiting
experiments
upon tail
dynes the
older
403
in Ref.3. available
values
rat
tendon
and whose tendon According
to
to the tendon rate
so that
The results
shown
modulus
are concerned) As a check
2.5 years.
cm -2 for
the
life-span.
the aging
the
words
properties
with
as described
seems that collagen
In other
load/extension
2.5 years
condition
(5.6
of their
as mechanical
smoothly
and it
at the age 50 days and 23 months
be completed
rat:
which
species,
human collagen.
we performed
good agreement
for
result
of the
as for
to age
in vitro
findings
would
plots
seems to have no connection
two rats
aged
longer
same curve
which
both
the collagen
interpretation
had been our
from
and dog lived
would
of this
that
curve listed
in Figs.2
the obtained and 3
and 3.6 x lo8 dynes
the modulus
agreement
is
cm -2
70
Vol. 79, No. 2, 1977
excellent (2.4
(4.8
BIOCHEMICAL
x lo’),
and the
of 54 year whereas
old
(3)
aging,
is work
more,
showed
authors
conversion
because
decrease
All properties
the
is reasonable
growth,
oxygen-dependent
background
of free
Finally, of connective the problem
maturity,
raised
be “toughened”-in
biological fact
most
could
forms
or without
oxygen.L changes
if
some of the (6,7)
throughout
are
is
reaction. the
aging
This
is
genetically by
In any case,
life. should
considered
a
of
are completed
tissues
It should be pointed out here that insolubility, and the magnitude of the stress and temperature of the fibre in an isometric melting experiment, co-valent cross-linking state of the fibre i.e. if the number and/or the thermal and mechanical cross-links increases.
404
with
at least
why the
effects
associated
with
constant
a cross-linking
has relevance.
all
concerned
the nature
work
not
categories
from the
Whatever with
in the
The other
solely
problem
that
system
of tissues.
consistent
reason
not be due to
age-related
fluid.
continues
aging
with
resulting
4 months
Further-
to more stable
determined
the present
process
after
be due to two distinct
tissue
reactions,
tendon
of insolubilisation
time
the
aging,
a marked
with
and repair
evidence
showed
upon oxygen.
that
a general
in vitro
dependent
place
genetically
is
which
by the
the aging
no obvious
is
for
cross-linking yet
is
there
tissue
took
could
in the
end result
rate
cross-links
mechanism
oxygen
its
also
suggest
maturation
continuous
the
changes
bonds
the
human tendon
of this
is
tissues
One is
mechanism,
old
curve
by 7 months
hypothesis.
the
in these
altered
melting
support
aldimine
of collagenous
formation,
determined
that
isometric
of “old”
above results
of cross-linking.
2.
at break
characteristics
fibres
of reducible
the
was not
(8) has shown that
collagen
there
stress
the
5.5 year
further
of reprecipitated the
for
the melting
Recent
this
for
that
tendon
curve
toward
of in vitro
finding
human wrist
the melting
alteration
the
agreement
x log). Our earlier
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
continue
to
to be undesirable.
mechanical strength attained before breakdown are all indices of the these parameters increase stability of the
Vol. 79, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The unavoidable oxygen-mediated reactions which we have postualted, provide an explanation for this problem.
References 1. Sinex, F.M. (1968) in "Treatise on Collagen", Gould B.S.(ed.) ~01.2, part B, pp.409-448, Academic Press, New York. 2. Rigby, B.J. (1967) Biochim. Biophys. Acta 140, 548-551. 3. Mitchell, T.W., and Rigby, B.J. (1975) Biochim. Biophys. Acta 393, 531-541. 4. Gross: J. (1963) Biochim, Biophys. Acta 71, 250-252. 5. Tanzer, M.L. (1973) Science 180, 561-566. 6. Bailey, A.J., Robins, S.P., and Balian, G. (1974) Nature 251, 105-109. 7. Bailey, A.J., and Shimokomaki, M.S. (1971) FEBSLetters, 16, 86-88. 8. Robins, S.P., and Bailey, A.J. (1977) Biochim. Biophys. Acta 492, 408-414.
405
would
