J. Biomedical Science and Engineering, 2010, 3, 1169-1174
doi:10.4236/jbise.2010.312152 Published Online December 2010 (http://www.SciRP.org/journal/jbise/ JBiSE
Published Online December 2010 in SciRes. http://www.scirp.org/journal/JBiSE
Different architectures of collagen fibrils enforce different
fibrillogenesis mechanisms
Mario Raspanti
Laboratory of Human Morphology, Insubria University 71 Via Monte Generoso, Varese, Italy.
Email: mario.raspanti@uninsubria.it
Received 25 October 2010; revised 29 October 2010; accepted 1 November 2010
According to current knowledge on collagen fibril-
logenesis, collagen fibrils are formed by a cooperative
process involving lateral fusion of small protofibrils.
Almost all the experimental research, however, was
carried out on tendon collagen, whose fibrils are
characterized by approximately straight subfibrils.
By contrast, in most tissues the collagen fibril sub-
units follow a helical course in which geometrical
constraints prevent lateral fusions, thereby implying
a different mechanism where collagen fibrils grow by
addition of individual microfibrils rather than by
lateral fusion of pre-assembled subfibrils. The proc-
ess at the origin of these fibrils may provide a simple,
automat ic explanation for the remarkable uniformity
in fibrils size observed in most tissues without re-
quiring the intervention of unknown mechanisms of
diameter control. Other mechanisms of growth con-
trol remain indispensable to terminate the fibril-
logenesis process in tendons and ligaments.
Keywords: Fibrillogenesis; Microfibrils; Collagen Fibril
Fibrillar collagens account for approximately a third of
the total body proteins. Their molecules, a coiled coil of
three individual polypeptide chains, ultimately form dis-
crete fibrils of indeterminate length and widely variable
diameter [1].
The diameter of collagen fibrils plays a central role in
the functional behaviour of the diverse connective tis-
sues [2,3], but the mechanisms controlling fibril size
remain unsettled. A number of different molecules and
factors have been shown to influence the collagen fibril
growth, including the relative content of collagen III [4]
and V [5], the terminal globular domains of the collagen
molecule (especially the N-terminal propeptide [6]),
cross-links [7], fibril-associated non-helical collagens (or
FACITs), COMP [8], small proteoglycans such as
decorin [9], fibromodulin [10], lumican [11] and bigli-
can [12], but also tenascin [13], perlecan [14], matrilin
[15] and thrombospondin [16]. Although there is some
evidence for most of these factors, none of them is by
itself entirely consistent with all the experimental obser-
vations. In particular none was able to explain why in
most tissues the fibril growth is very effectively con-
trolled so that the collagen fibrils size is highly uniform
and distributed along a narrow Gaussian curve, while in
other, and often adjoining, tissues the collagen fibrils are
large and inhomogeneous with a distinctive multimodal
distribution. The difference between these two classes is
so great that it may seem not unreasonable to hypothe-
size that two entirely different mechanisms are at work
in different tissues.
It is important to bear in mind that these two classes
of fibrils represent two mutually exclusive subfibrillar
architectures [17-19]. Historically, research on fibril ar-
chitecture and fibrillogenesis has been carried out almost
exclusively on the large variable fibrils of tendons,
whose subfibrils run almost parallel to the fibril axis.
These fibrils come into being by means of a cooperative
mechanism involving the lateral fusion of smaller proto-
fibrils [20]: the process has been directly observed in
vivo, and in vitro even mature fibrils of type I collagen
retain the ability to fuse laterally in thick clumps unless
fusion is inhibited by small proteoglycans [21].
Much less attention has been paid to the smaller uni-
modal fibrils. Corneal fibrils have been painstakingly
demonstrated to be made of discrete subunits, corre-
sponding in size to the microfibrils of Smith [22] and
laid out in concentric layers [23,24] where they wind in
a right-handed helix. In each layer the microfibrils wind
at a constant angle of 15 to 17 degrees with respect to
the fibril axis, the precise measurement depending on the
technique used. As a consequence, the axial D-period is
reduced from the usual 67 nm to approximately 64 nm
M. Raspanti / J. Biomedical Science and Engineering 3 (2010) 1169-1174
(64 67cos(17º)) [25], and the tilt-caused shear brings
adjoining microfibrils in such a position to allow the
formation of a peculiar covalent cross-link involving a
histidine residue [26]. The diameter of these “helical”
fibrils varies from tissue to tissue but remains extremely
uniform in each location. Other tissues with small het-
erogeneous fibrils (such as blood vessels and nerve
sheaths) have been less studied, but the facts that they all
share an identical winding angle, the same shortened
D-period and an equally narrow diameter distribution are
strongly suggestive of an identical substructure. This
subfibrillar architecture, defined as a constant angle he-
lix and originally entertained in 1989 [19], is not easy to
visualize: the pitch of the helix that each subfibril de-
scribes around the fibril axis is 2πrtan(/2-) (where
is the angle with respect to the fibril axis) and therefore,
all other factors being constant, it is variable with the
On the other hand, this design automatically guaran-
tees that all the microfibrils have an identical axi-
ally-projected length of 64 nm (an essential condition for
the distinctive banding pattern of collagen to appear).
The only alternative model, the constant pitch helix, re-
quires axial subunits to be compressed and peripheral
ones stretched in order to be consistent with ultrastruc-
tural data [19]. Moreover, because of its layered struc-
ture, the constant angle helix has the additional advan-
tage of being consistent with the discrete distribution of
diameter values reported in some tissues [2].
This structure has another simple but critical conse-
quence which, to the best of our knowledge, has not
been noticed so far: such helical fibrils cannot possibly
undergo lateral fusion since the adjoining microfibrils of
two parallel fibrils always wind in opposite directions
(Figure 1). This, of course, also holds true for antiparal-
lel fibrils, which would be unable to merge anyway. It is
an everyday observation that wire ropes are made of
strands that never merge: for a helically-wound fibril to
merge with another similar fibril, it would have to be
entirely unwound and rewound, a process made unlikely
by the substantial variation of free energy it implies.
In other words, while the large heterogenous size of
tendon collagen fibrils may emerge as the result of a
random lateral fusion of protofibrils taking place during
the first phases of fibrillogenesis (until this process is
inhibited by fibril-bound proteogycans and/or by some
other mechanism), the slender fibrils of cornea, blood
vessels and sheaths must simply precipitate from a su-
persaturated solution of collagen molecules. Under these
conditions each growing fibril competes with its
neighbours for the available subunits until all these have
been depleted. The extracellular environment being the
same for all forming fibrils in a given location, they all
Figure 1. The adjoining microfibrils of two parallel fibrils run
in opposite directions (red arrows) preventing any lateral fu-
sion. Model developed on Alias|Wavefront Maya 6.0.1 running
under IRIX on a Silicon Graphics Fuel R14000/600.
end up of a similar size without requiring (at least in
principle) any external limiting factor. Again, this phe-
nomenon is readily observed in everyday life: for in-
stance in snowflakes or hailstones which, albeit variable
from place to place, tend to have the same size in the
same location.
A simple program was developed to test this hypothesis.
The program essentially tries to obtain a graphical simu-
lation of the cross-section of a bundle of cylindrical fi-
brils. Each individual pixel represents a 4-nm microfibril;
the program typically manages a 500 x 500 pixel arena,
corresponding to a 2000 2000 nm field, which is
enough to create a few hundred fibrils, which in turn can
represent a significant sample.
The program can be outlined as follows:
1) A new microfibril is created in a random position
Copyright © 2010 SciRes. JBiSE
M. Raspanti / J. Biomedical Science and Engineering 3 (2010) 1169-1174
Copyright © 2010 SciRes.
2) If no fibril lies within a given “capture distance” (a
user-defined value set by the Field parameter – see Fig-
ure 2), then the microfibril itself becomes a new fibril;
else the microfibril coalesces with its nearest fibril,
whose radius is recomputed accordingly.
3) A test is made to check if the fibril, which has now
grown larger, collides with any other fibril. If a collision
is found:
4) If fibril merging is enabled (via the Merge switch)
then the two fibrils merge into a new one, whose radius
and position are recomputed; else the colliding fibril is
simply displaced.
5) If fibril merging is enabled, a random function
(whose probability increases with the fibril size) can
block the fibril, preventing other subsequent fusions.
6) Point (3) is recursively repeated until no further
collisions are found.
The whole process is iterated up to the desired volume
fraction (set by the Fraction parameter).
As in any simulation, a few arbitrary assumptions had
to be made: Figure 2. The graphical output of the fibrillogenesis simulation
program. When fibril fusion is enabled, the microfibrils form
fibrils with a widely variable distribution of diameters, ranging
from 4 nm to over 450 nm as observed in tendons and liga-
1) In the real world the capture distance is likely to
depend on a complex interplay of several factors which
are impossible to quantify precisely. One of them is the
creation rate of the microfibrils (if the rate is low enough
there is more time for the Brownian motion to bring new
microfibrils into contact with a preexisting fibril). In
software this parameter was empirically set to a fraction
of a collagen molecule length.
2) The volume fraction of collagen is also variable
from tissue to tissue. In our simulation this parameter is
not really important, since the fibrils in excess simply
exit the simulation arena and are lost, so it was set to
3) The termination condition of point (5) was intro-
duced to prevent fusion-enabled fibrils merging into a
single huge unit, that is precisely what happens in vitro
when no control factors are present [21].
Even under these relatively crude conditions the pictures
show a clear similarity with the actual micrographs. If
the lateral fusion is allowed then the process leads to the
appearance of widely heterogenous aggregates (Figure 2)
quite similar to the fibrils found in tendons and liga-
If fibril fusion is precluded, the simulation yields a
field of remarkably uniform fibrils (Figure 3). In this
case the process even mimics the apparent alignment of
fibrils in rows, often visible in electron micrographs
(compare these pictures with Figure 5 of Ref. [1]).
Figure 3. When the lateral fusion is disabled, all other pa-
rameters remaining constant, the result is a field of remarkably
uniform fibrils with a diameter centered around 100 nm in a
narrow distribution. The simulation software was written in
tandard Java 2 and runs on the same workstation as Figure 1.
It must be stressed that the software is intended to just s
M. Raspanti / J. Biomedical Science and Engineering 3 (2010) 1169-1174
Figure 4. Exploring the parameter space in our simulation changes the average diameters but does not alter their distribution. The
two panels above show the diameter distribution (in nm) obtained when the lateral fusion is not allowed: changing the two
user-defined parameters always produces a family of Gaussian curves, centered on different values but with a similar shape. The
two panels below depict the multi-peak distribution obtained under the same conditions if the lateral fusion is enabled.
reveal a general trend, and does not claim to be a com-
prehensive reconstruction of the fibrillogenesis process.
It is noteworthy, however, that varying all the parameters
influenced the average diameter of fibrils but did not
alter the diameter distribution: always a smooth Gaus-
sian in one case, a multi-peak range in the other (Figure
Our results suggest that the remarkable uniformity in
size that collagen fibrils show in some tissues does not
require the introduction of some unknown, stringent
mechanism of diameter control, but emerges spontane-
ously as the result of a simple structural constraint. The
most effective mechanism of growth control is one that
does not depend on external interventions: the helical
substructure of these fibrils is both a necessary and a
sufficient condition for their diameter uniformity.
These results do not imply than propeptides or pro-
teoglycans or other molecules have no part in limiting
the lateral accretion of the fibrils; quite the opposite. The
role, for instance, of SLRPs in this process is undeniable
[21], and even in our simulation a termination condition
had to be introduced if lateral fusion was allowed.
Rather, these results imply that the absence of external
growth-limiting factors may affect different fibrils (and
hence different tissues) in different ways. In tissues such
as tendons whose fibrils have straight microfibrils, the
proteoglycans (and/or propeptides or other molecules)
are necessary to terminate lateral fibril aggregation
whereas in tissues such as cornea, nerve sheaths and
interstitial collagen of parenchymatous organs whose
fibrils have helical microfibrils they are not. Therefore a
DCN -/- organism, for instance, can be expected to show
fibrillar alterations in tendons but not in cornea. And this
is exactly what is found in experimental observations
For historical and technical reasons connective tissue
research is so deeply entrenched in the study of tendon
and bone that the mere existence of fibrils with helical
subunits took many years to emerge. Although inde-
pendently confirmed by freeze-etching [17], cross-links
analysis [26], SEM and AFM [27], electron tomography
[23] and X-ray diffraction [24], the evidence has yet to
Copyright © 2010 SciRes. JBiSE
M. Raspanti / J. Biomedical Science and Engineering 3 (2010) 1169-1174 1173
find its way into histology textbooks. We must take care
not to repeat the original sin of studying only tendons,
and then ascribe what we found here to all other tissues.
Fibrils with helical subfibrils are the most common
among our different tissues. Our data indicate that the
current knowledge on the fibrillogenesis process, all
gained on tendons, does not apply to these fibrils, and
that new research is required.
The next primary objective will be the identification
of the molecular switch controlling the initiation of one
or the other subfibrillar architecture, and which has so
far eluded detection. Fibrillogenesis is a delicate process
which can be influenced by myriad causes, some of
which do not even remain to make up the final product.
Perhaps the most promising candidate appears now
type V collagen. Almost since its discovery the type V
has associated with the control of fibril diameter [28],
and more recently it seems to be essential for fibril for-
mation in vivo: homozygote engineered organisms lack-
ing type V die at embryonic day 10 of cardiovascular
failure, while heterozygotes are viable but lack about
half their collagen fibrils [29].
It seems therefore not unreasonable to hypothesize
that type V collagen is involved in the formation of
slender, uniform fibrils in cornea, blood vessels and
sheaths, but obviously not in tendon and ligaments
where it appears only in traces. Further research is un-
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