MECHANICAL PROPERTIES OF SINGLE TYPE II COLLAGEN MOLECULE
*Sun, Y; **Luo, Z; +*An, K
+*Biomechanics Laboratory, Mayo Clinic, Rochester, MN 55905. 507-538-1717, Fax: 507-284-5392, [email protected]
Introduction
Among nineteen types of collagen identified in a wide range of vertebrate
tissues, type II collagen is one of the most common collagens and forms the
principle fibrillar component of cartilage. Alterations of type II collagen are
responsible for many joint diseases, such as osteoarthritis. Previous
biomechanical studies suggested that mechanical properties of cartilage at the
gross level are related to the contents of molecules and assembly of the lower
level structures. To date, little has been known regarding the mechanical
properties of cartilage below the gross level. By using the-state-of-art
nanotechnology, this study was designed to experimentally measure the
mechanical properties of single type II collagen molecules.
Methods
The experiment was performed by the use of optical tweezers/interferometer
system developed in our laboratory[1]. Human type II procollagen was
prepared from cell culture. It was reduced with dithiothreitol (Sigma) and
biotinylated using EZ-Link PEO-maleimide activated biotin (Pierce).
Biotinylated type II procollagen was adhered to the streptavidin-coated
polystyrene beads (diameter 2.2 um, Spherotech Inc.), which was connected
with biotin-coated polystyrene beads (diameter 6.7 um, Spherotech Inc.).
Under the microscope, a streptavidin-coated polystyrene bead (diameter 4.1
um) was trapped by laser tweezers and tapped against the bead covered with
type II procollagen. Connection was established between two beads through
type II procollagen. The molecules were stretched by moving the large bead
away from the laser beam center with a fixed rate (73 nm/s) in a solution of 25
mM KCl, 1 mM EGTA, 0.2% Tween 20, 0.1% casein and 25 mM HEPES pH
7.4 (Figure 1). Totally, forty-five molecules were tested. The forcedeformation relationship was fitted into a worm-like chain elastic model [2].
Both the contour length and the persistence length (a measure of molecule
bending rigidity) of the molecule was then determined. The elastic modulus
was further estimated from the persistence length [3].
Persistence length is directly proportional to the intrinsic elastic constant of
the polymer. The elastic modulus of type II collagen of 270 MPa was smaller
than the elastic modulus of type I collagen (350 MPa). It reflects the
difference between the mechanical function of tissues consisting of these two
types of collagen.
The present study sheds light on the mechanical behaviors of single type II
collagen molecule, which is essential in studying the interaction between cells
and surrounding extracellular matrix; the relationship between the structures
and mechanical properties of cartilage; and the etiology of cartilage
degeneration and mechanism of regeneration.
Cover Glass
Procollagen
Trap Center
Cover Glass
XY Stage
Laser Light
Fig. 1. Stretching a procollagen II molecule with an optical tweezers.
14
Results
The results demonstrated highly nonlinear force-extension curves for type II
collagen molecule (Figure 2). The molecule was stretched by a force (< 3 pN)
to near their contour length. The force increased sharply when the molecules
were stretched to their full contour length. Both the persistence length and the
contour length were simultaneously determined by fitting the force-extension
measurement into an entropic worm-like chain model. The persistence length
of 11.2±8.4 nm was obtained for single collagen II molecule. The molecule
length of collagen II was 295.8±31.9 nm. The elastic modulus of type II
collagen estimated from the persistence length was 270±198 MPa.
Discussion
Mechanical properties of single type II collagen molecules were measured in
this study. To our knowledge, this was the first direct measurement of this
type of collagen. The measured contour length of 295.8 nm in current study
compared favorably with that of 300 nm reported in the literature. This
agreement suggested that the entropic worm-like chain model could apply to
collagen molecules as other biopolymers.
Persistence length is the standard measure of polymer rigidity and flexibility.
The persistence length of type II collagen is about one twenty-seventh of its
contour length. It is slightly smaller than that of type I collagen (14.5 nm) and
is much shorter than that of DNA (50 nm), F-actin (1.8 microns) and
microtube (5200 microns). This suggests that a single type II collagen
molecule is very flexible rather than a rigid rod-like molecule under
physiological conditions.
12
experimental data
theoretical model
Force (pN)
10
8
6
4
2
0
-2
0
50
100
150
200
250
300
350
Extension (nm)
Fig. 2. The force-extension curve of a single collagen II.
References:
1. Luo, Z. P., Bolander, M. E., and An, K. N., Biochem. Biophys. Res.
Commun., 232: 251-254, 1997.
2. Bustamante, C., Marko, J. F., Siggia, E. D., and Smith, S., Science,
265: 1599-1600, 1994.
3. Baumann, C. G., Smith, S. B., Bloomfield, V. A. and Bustamante, C.,
Proc. Natl. Acad. Sci. USA, 94: 6185-6190, 1997.
Acknowledgements
This study was supported by grants from NIH-NIAMS (R01 AR44497),
Arthritis Foundation and Mayo Foundation.
**Department of Orthopedic Surgery, Baylor Collage of Medicine, Houston,
Texas 77030.
48th Annual Meeting of the Orthopaedic Research Society
Paper No: 0082