Atomic force microscopy findings are twisted
Study belies long-held beliefs about collagen fibril formation
Sometimes it is necessary to revisit established ideas that are widely
circulated as dogma. Investigators with University College London affirmed this
recently when studying a variety of collagen-based samples — including rat-tail
extracts, engineered collagen sheets used in implants, and historical parchments
made from animal skin — with atomic force microscopy.
“The idea was to review the morphology of
the collagen itself, but at the nanoscale,” explained researcher Laurent Bozec.
“After a few images, we found the well-known D-banding — a consistent
periodicity along the fibril — and wanted to understand where that characteristic
banding was coming from.”
Researchers have used atomic force microscopy (AFM) to uncover the morphology of collagen fibrils
— as seen in the optical image in (a). For decades, investigators have subscribed
to a model of fibril structure that explains the periodicity in some fibrils as
a staggered repeat of collagen molecules, but researchers at University College
London have confirmed a twisted, ropelike structure. Here, AFM images of collagen
fibrils from bovine digital tendons (c) are contrasted with those from rat tails
(d), which exhibit the same banding but which are much straighter . Panel (b) is
a lower-magnification AFM image of the tendon collagen fibrils. Images reprinted
with permission of Biophysical Journal.
The problem was that most everyone
who studies such things — including first-year medical students — already
knows where it comes from. Or they think they do, anyway. Hodge and Petruska proposed
a model of collagen fibril formation more than 40 years ago and, although it leaves
open several structural and functional questions, this model remains more or less
the final word on the topic. “We didn’t want to take any early studies
for granted,” Bozec explained, “so we had to come up with our own model
and see where it fit in the current literature.”
The accepted wisdom suggests that collagen
fibrils are formed through staggered repeats of individual molecules — leading
to the consistent periodicity along the fibrils. This model is essentially two-dimensional,
Other investigations have sought to
address the resulting deficiencies — for example, one study posited a layered,
spiral arrangement of collagen molecules. But studies have failed to explain a number
of characteristics, including the preservation of D-banding regardless of fibril
diameter and the fact that fibrils up to 10 nm in diameter form into fibrils of
greater diameter — and thus into macroscale objects such as tendons.
Mechanical modeling further supported
the ropelike structure of the fibrils. Image (a) shows patterns for a single strand
(top) and the corresponding two-ply (bottom), for a fibril with no intrinsic twist.
Image (b) shows a pattern for a six-ply fibril with an intrinsic twist along with
a corresponding AFM image. Such modeling helped make experimentally verifiable predictions
about the fibrils’ behavior.
More recent studies have revealed a
possible ropelike structure to the fibrils. Unlike the theories from the earlier
studies, this would satisfy an established dictum in biology: A tissue’s form
follows its function. Hence, the twisted rope features of the fibrils — the
source of periodicity — may be crimps resulting from the relaxation of subcomponents
of the fibrils that contribute in some way to their mechanical features.
In a Biophysical Journal paper
published online Oct. 6, the University College London researchers reported a study
in which they used atomic force microscopy to confirm the ropelike model of fibril
periodicity. They sought to understand the architecture of the collagen fibril by
trying to open the fibril itself. “Other studies have shown some degree of
success by looking at the different stages of the fibrillogenesis and deducing the
overall architecture,” Bozec said. “We took an intact fibril and looked
at it under a microscope as it unravels.”
“Scientists believe the ‘messy’
structure they sometimes see is just wrong,” he added. “We decided to
look deeper inside those abnormalities and discovered clues that helped us to understand
the fibrillar architecture.”
To this end, they used atomic force
microscopes made by Veeco Instruments Inc. of Santa Barbara, Calif., and by JPK
Nanowizard AG of Berlin to record topologic (height) and error signal (deflection)
images of native bovine digital tendon and rat-tail (or flexor digitalis) tendon
collagen fibrils. They operated the microscopes in contact mode using cantilever
tips also made by Veeco Instruments.
The images provided very strong evidence
of a ropelike structure — suggesting that many investigators must re-evaluate
their existing structural data sets, as well as their long-held beliefs about periodicity.
The new model also offers experimentally verifiable predictions. For example, if
fibrils’ ropelike structures are a response to their mechanical needs, then
fibrils exposed to tension should be more inclined to twisting during formation.
All of which has important implications
for understanding the biomechanics of the human body, and clinical implications
in particular. For example, investigators are working to develop collagen-based
scaffolds for tissue-engineering applications. The insights afforded by the present
study suggest that twisted fibrils should be used for such scaffolds in arteries
and other mechanically stressed locations, because the fibrils are essentially designed
to meet the needs of scaffolds in these locations.
Questions remain, however. Regardless
of topology or dimensions, the fibrils had one thing in common: All exhibited D-banding
periodicity of 69.6 ±2.9 nm.
What accounts for the apparent disconnect
between topology and D-banding periodicity? And how do single molecules come together
to form this periodicity independent of fibril diameter? To address these and
other questions, the researchers are working to refine the model and to look deeper
inside the fibril with atomic force microscopy. The model is currently incomplete,
Bozec said, because it simplifies the triple-helical structure of the molecule to
that of a patterned cylinder. “We need to use a more realistic base unit for
an even closer-to-reality model.”
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