JOURNAL OF STRUCTURAL BIOLOGY 122, 119 122 (1997)
ARTICLE NO. SB983966
Fibrillar Structure and Mechanical Properties of Collagen
Peter Fratzl,1 Klaus Misof, and Ivo Zizak
Materials Physics Institute and Ludwig-Boltzmann Institute of Osteology, University of Wien, Strudlhofgasse 4, A-1090 Wien, Austria
Gert Rapp
European Molecular Biology Laboratory Outstation, Notkestrasse 85, 22603 Hamburg, Germany
Heinz Amenitsch
Institute of Biophysics and X-Ray Structure Research, Austrian Academy of Sciences, Steyrerg. 17, A-8010 Graz, Austria
and
Sigrid Bernstorff
Sincrotrone Trieste, Strada Statale 14 - Km. 163.5, 34012, Basovizza, Trieste, Italy
Received November 13, 1997
mined by the collagen structure. Tendons are built of
Collagen type I is among the most important
parallel fibrils which are themselves assemblies of
stress-carrying protein structures in mammals. De-
parallel collagen molecules. There is still no com-
spite their importance for the outstanding mechani-
plete understanding of the relation between stress-
cal properties of this tissue, there is still a lack of
induced changes in the structure and the specific
understanding of the processes that lead to the
shape of the stress/strain curve of collagen.
specific shape of the stress strain curve of collagen.
Typically, the stress/strain curve of collagen from
Recent in situ synchrotron X-ray scattering experi-
tendon can be subdivided into several regions (Vin-
ments suggest that several different processes could
cent, 1990), as outlined in Fig. 1. The region of small
dominate depending on the amount of strain. While
at small strains there is a straightening of kinks in strains (  toe  region) corresponds to the removal of a
the collagen structure, first at the fibrillar then at
macroscopic crimp in the collagen fibrils, visible in
the molecular level, higher strains lead to molecular
the light microscope (Diamant et al., 1972). At larger
gliding within the fibrils and ultimately to a disrup-
strains (in the   heel  and the   linear  region of the
tion of the fibril structure. Moreover, it was ob-
stress/strain curve, see Fig. 1), there is no further
served that the strain within collagen fibrils is
structural change visible in the light microscope.
always considerably smaller than in the whole ten-
Hence, the processes affecting the collagen structure
don. This phenomenon is still very poorly under-
occur in the submicrometer range and can be investi-
stood but points toward the existence of additional
gated by (synchrotron) X-ray scattering.
gliding processes occurring at the interfibrillar
level. 1997 Academic Press
HEEL REGION OF THE STRESS/STRAIN CURVE
INTRODUCTION At strains typically beyond 3%, the stiffness of rat
tail tendon increases considerably with the exten-
The outstanding mechanical properties of collagen-
sion (heel region, Fig. 1). In a recent synchrotron
rich tissues like, e.g., tendons, are largely deter-
X-ray scattering experiment, Misof et al. (1997a)
have studied the structural changes occurring in this
part of the stress/strain curve. It was observed that
1
To whom correspondence and reprint requests should be
the intensity of the diffuse equatorial scattering,
addressed at present address: Erich Schmid Institute of the
which is due to the lateral arrangement of the
Austrian Academy of Sciences and University of Leoben, Jahnstr.12,
A-8700 Leoben, Austria. E-mail: [e-mail]. collagen molecules inside the fibrils (Fratzl et al.,
119
1047-8477/97 $25.00
Copyright 1997 by Academic Press
All rights of reproduction in any form reserved.
120 FRATZL ET AL.
(see Fig. 1). The model also implies a linear relation
between strain and degree of lateral order, which
was observed experimentally.
LINEAR REGION OF THE STRESS/STRAIN CURVE
When collagen is stretched beyond the heel region
of the stress/strain curve, most kinks are straight-
ened and no further extension is possible by the
entropic mechanism described above. Therefore, some
other process must prevail in the linear region of the
stress/strain curve. The most likely processes are a
stretching of the collagen triple-helices or of the
cross-links between the helices, implying a side by
side gliding of neighboring molecules. This process
has already been studied in the mid-eighties by use
of synchrotron radiation diffraction experiments
FIG. 1. Typical stress strain curve of a rat tail tendon. In the
(Mosler et al., 1985; Folkhard et al., 1986). In these
toe region, where the tendon can be extended with very little force,
experiments, a strain-induced change in the struc-
a macroscopic crimp of the fibrils with a typical period in the order
of 100 µm is removed (Diamant et al., 1972). This can be
ture factor of the axial diffraction maximums was
visualized using polarized light (a). Further structural changes
observed. In particular, the second order maximum
occur at the fibrillar level (b). The heel region may correspond to a
increased with respect to the third order, when the
straightening of molecular kinks in the gap (Misof et al., 1997a)
tendon was stretched. This was a clear indication
and the linear region to a gliding of molecules (Folkhart et al.,
1986). The most recent synchrotron diffraction data suggest that a
that stretching increased the length of the gap
disruption of the fibrillar structure starts with an increased
region with respect to the length of the overlap
fuzziness of the gap/overlap interface (see schematic picture, top
region, implying a considerable gliding of neighbor-
right).
ing molecules (Folkhard et al., 1986).
In a very recent experiment, we have revisited this
problem by measuring the intensities of the meridi-
1993; Hulmes et al., 1995), increased linearly with
onal reflections of wet rat tail tendons and control-
the strain. This was interpreted as a reduction of the
ling the external stress and strain by means of the
disorder in the lateral molecular packing within
apparatus described in Misof et al. (1997a). The
fibrils, resulting from the straightening of kinks in
experiments were carried out at the SAXS beamline
the collagen molecules.
of the synchrotron source ELETTRA in Trieste
Indeed, kinks are thought to occur within the gap
(Amenitsch et al., 1995). The data were collected
region of the collagen fibril structure. In particular, a
using an X-ray CCD camera (AXS, Karlsruhe). This
recent refinement of the collagen fibril packing struc-
two-dimensional data collection allowed an integra-
ture (Wess et al., 1998) points toward the existence of
tion of the peak intensities accounting for the fact
kinks. They might occur in the gap region of the
that the unit cell of the collagen structure is tilted by
collagen fibrils because of the greater flexibility of
a few degrees with respect to the fibril axis (Wess,
collagen molecules due, first, to lower levels of
1998), which leads to a splitting of higher order
proline and hydroxyproline on the collagen chain
meridional peaks. Both the applied stress and the
(Fraser and Trus, 1986) and, second, to the reduced
overall strain on the tendon were recorded during
packing density as compared to the overlap region
the experiment.
(Fraser et al, 1983). Moreover, considerable azi-
Figure 2 shows the evolution of peak intensities as
muthal and lateral flexibility of collagen molecules
a function of the D-period during a typical stretching
had been demonstrated in NMR measurements (Je-
experiment. There are two remarkable effects:
linski et al., 1980).
1. Odd and even orders behave in a qualitatively
The model outlined in Misof et al. (1997a) assumes
different way. While odd orders always decrease with
that spontaneously occurring molecular kinks (that
strain, the lower even orders first increase and then
is, kinks appearing by thermal activation) lead to an
decrease (Fig. 2). This means that, in particular, the
increased disorder and, hence, entropy of the gap
ratio of second to third order increases drastically
region. The straightening of the kinks would lead,
therefore, to an elongation of the fibril and to a during the stretching process, an effect that has been
reduction in entropy which provides the force acting observed before (Mosler et al., 1985). In normal
against the elongation. This entropic force is increas- tendon this ratio is typically very small (Brodsky et
ing when the number of kinks decreases leading to al., 1982) and its increase can be interpreted as the
the typical upwards curvature of stress/strain curve growth of the gap in comparison to the overlap region
FIBRILLAR STRUCTURE AND MECHANICAL PROPERTIES OF COLLAGEN 121
means that the amount of disorder in the axial
staggering increases upon stretching. This was, how-
ever, not accompanied by a broadening of the axial
peaks during the stretching experiment.
A simple way to explain this observation is the
assumption that the interface between gap and
overlap region is getting increasingly fuzzy, as shown
schematically in the top right image of Fig. 1. This
may occur when the relative gliding of the molecules
is not exactly the same for each nearest neighbor
pair. Under this assumption, the axial projection of
the electron density would be smeared by a distribu-
tion function. Calling it P(r) and the electron density
without the smearing g0(r), the resulting electron
density along the fibril would be the convolution of P
and g0, P g0. Hence, the intensities of the axial
peaks would be determined by the Fourier-trans-
form squared of P g0, that is, the product of the
squared Fourier transforms of P and of g0. Conse-
quently, if P is, e.g., a Gaussian with width w, then
the axial peak intensities are multiplied by the
squared Fourier transform of P, which is a Gaussian
FIG. 2. Evolution of the meridional peak intensities of rat tail
of a width proportional to 1/w. Hence, the stronger
tendon under tensile stress, the length of the tendon being
the smearing, the larger the damping of higher order
increased at a constant rate. The intensities are shown as a
function of the D-period and were normalized to their value at D peaks. As a result, the intensities of all axial reflec-
66.85 nm, which was the D-period at the beginning of the
tions will eventually decrease with increasing strain,
stretching process. The odd and even orders are shown separately
higher orders faster than the lower ones. This may
in the upper and lower panel, respectively. Some of the weak
explain, at least qualitatively, the effects observed in
orders (4 and, particularly, 8) are affected by large statistical
Fig. 2.
errors. Shortly before the tendons started to break, partial
relaxation of the D-period was occasionally observed (like between Finally, the relation between the increase of the
the ninth and the tenth point in the graph, around D 68.6 nm).
collagen D-period (that is, the strain at the fibrillar
This is most probably the result of a partial stress release due to
level) and the macroscopic strain of the specimen is
the failure of some of the collagen fibrils in the assembly.
shown in Fig. 3. In the linear region of the stress/
strain curve, the D-period increased by about 40% of
the macroscopic strain. This implies that not all the
of the fibril, which indicates a gliding of neighboring
elongation of the tendon is due to a stretching of the
molecules with respect to each other.
fibrils (Sasaki and Odajima, 1996), and suggests
The Hodge Petruska staggering (1963) implies
that some of the elongation of the tendon is due to a
that the gap length and the molecular length add up
relative movement of entire fibrils. At this point we
to 5 D. Therefore, calling and the relative
D M
can only speculate about possible mechanisms, but it
increase of the D-period and of the length of the
is not unlikely that an interfibrillar gliding is medi-
triple-helical molecule,
ated by a highly viscous interfibrillar substance
containing water and proteoglycans.
5 (5 ) ,
0 D 0 M
OPEN QUESTIONS
where is the ratio of the gap length to the D-period,
with being its value at the beginning of the Recent synchrotron X-ray scattering experiments
0
stretching process. Since the molecule is consider- have revealed drastic changes in the molecular
ably stiffer than the fibril (Sasaki and Odajima, packing of collagen fibrils under strain. While at low
1996), increases more slowly with the applied strains a straightening of molecular kinks seems to
M
stress than , which means that , the fraction of dominate, molecular gliding is observed at large
D
the D-period occupied by the gap, increases with strains, leading to an increasingly irregular exten-
external stress. This may, in turn, explain qualita- sion and ultimately to a disruption of the fibrillar
tively the experimentally observed increase of sec- structure. The mechanisms are summarized in Fig.
ond order peak in Fig. 2. 1a, which shows the macroscopic effects occurring in
There is a systematic trend that higher reflections the toe-region of the stress/strain curve. Figure 1b
decrease more rapidly than lower ones. This is shows mechanisms at the fibrillar level. First, a
particularly visible for the even orders (Fig. 2) and it straightening of molecules and then an increase of
122 FRATZL ET AL.
ever, suggest a considerable importance, since only
40% of the strain on the rat tail tendon is actually
transmitted to the fibrils.
This work has been supported by the Fonds zur Förderung der
Wissenschaftlichen Forschung (P11762-PHY).
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