Oxygen participation in the in vivo and in vitro aging of collagen fibres

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

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Vol. 79, No. 2, 1977

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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