2204 Biophysical Journal Volume 94 March 2008 2204 2211
Mechanical Properties of Native and Cross-linked Type I Collagen Fibrils
Lanti Yang,* Kees O. van der Werf,y Carel F. C. Fitié,* Martin L. Bennink,y Pieter J. Dijkstra,* and Jan Feijen*
*Polymer Chemistry and Biomaterials, Faculty of Science and Technology and Institute for Biomedical Technology, and
y
Biophysical Engineering, Faculty of Science and Technology and MESA1 Institute for Nanotechnology, University of Twente,
Enschede, The Netherlands
ABSTRACT Micromechanical bending experiments using atomic force microscopy were performed to study the mechanical
properties of native and carbodiimide-cross-linked single collagen fibrils. Fibrils obtained from a suspension of insoluble
collagen type I isolated from bovine Achilles tendon were deposited on a glass substrate containing microchannels. Force-
displacement curves recorded at multiple positions along the collagen fibril were used to assess the bending modulus. By fitting
the slope of the force-displacement curves recorded at ambient conditions to a model describing the bending of a rod, bending
moduli ranging from 1.0 GPa to 3.9 GPa were determined. From a model for anisotropic materials, the shear modulus of the
fibril is calculated to be 33 6 2 MPa at ambient conditions. When fibrils are immersed in phosphate-buffered saline, their
bending and shear modulus decrease to 0.07 0.17 GPa and 2.9 6 0.3 MPa, respectively. The two orders of magnitude lower
shear modulus compared with the Young s modulus confirms the mechanical anisotropy of the collagen single fibrils. Cross-
linking the collagen fibrils with a water-soluble carbodiimide did not significantly affect the bending modulus. The shear modulus
of these fibrils, however, changed to 74 6 7 MPa at ambient conditions and to 3.4 6 0.2 MPa in phosphate-buffered saline.
INTRODUCTION
Collagen, the most abundant protein in the human body, tendon, bone, and cartilage has been studied with different
provides structural stability and strength to various tissues. techniques (6 9) and using theoretical modeling (10 12). It
About 25 types of collagen have been identified, of which is suggested that the mechanical anisotropy at the fibril level
collagen type I is the major component of the fibrous struc- and the highly ordered parallel packing of fibrils result in
ture of skin, tendon, and bone in the human body (1). mechanical anisotropy of most tissues (13,14). However,
Studies on the collagen type I structure have shown a current mechanical approaches cannot easily separate the
complex hierarchical arrangement of collagen subunits. In contribution of mechanical anisotropy as a result of the hier-
this hierarchical arrangement, it is widely accepted that five archical arrangement of collagen molecules in the fibril and/or
tropocollagen molecules assemble into microfibrils (2 4). Of parallel packing of the fibrils.
the various hypothesized models, the compressed microfibril Efforts have been made to determine the mechanical
model (2) and the supertwisted right-handed microfibril properties of collagen single fibrils using different micro-
model (3) most closely fit the x-ray diffraction data. Recently, mechanical techniques. Graham et al. (15) stretched in vitro-
the structure of microfibrils has been visualized using atomic assembled type I collagen fibrils obtained from human
force microscopy (AFM) imaging (5). These microfibrils fibroblasts using AFM and obtained a Young s modulus of
aggregate in lateral and longitudinal direction to form fibrils. 32 MPa. Eppell et al. (16) studied the stress-strain relation of
The collagen fibrils with diameters between 10 and 500 nm single type I collagen fibrils isolated from the sea cucumber
further assemble into fibers that become part of the structural and found a Young s modulus of 550 MPa in the hydrated
skeleton of tissues. Because of the highly organized mode of state. Also, in our lab, we used a home-built AFM system to
self-assembly, a single collagen fibril is regarded as homoge- perform tensile tests on single collagen type I fibrils isolated
neous, which means it has the same composition throughout from bovine Achilles tendon (17). A Young s modulus of 5 6
the fibril. However, the alignment of collagen molecules and 2 GPa for dry collagen type I fibrils was found, and when these
microfibrils in the longitudinal fibril direction may induce fibrils were immersed in phosphate-buffered saline (PBS), the
mechanical anisotropy of the single collagen fibrils. The Young s moduli ranged from 0.2 to 0.5 GPa. Very recently,
packing of these structural components and the organization the reduced modulus of collagen single fibrils isolated from rat
of the collagen fibrous structure are crucial to the mechanical tail tendons was determined by nanoindentation using AFM
function of tissues. Mechanical anisotropy of tissues such as (18) in air at room temperature and ranged from 5 to 11 GPa.
These results support the hypothesis that the anisotropy of
collagen results from the alignment of subfibrils along the fibril
Submitted August 19, 2007, and accepted for publication October 17, 2007.
axis. However, current methods to investigate the mechanical
Address reprint requests to Jan Feijen, Polymer Chemistry and Biomate-
properties of single collagen fibrils are limited as no shear
rials, Faculty of Science and Technology and Institute for Biomedical
related mechanical properties are measured.
Technology (BMTI), University of Twente, PO Box 217, 7500 AE,
Recently an AFM-based three-point bending technique has
Enschede, The Netherlands. Tel.: 31-53-4892968; Fax: 31-53-4892155;
E-mail: J.Feijen@utwente.nl. been developed by different groups to measure the mechanical
Editor: Thomas Schmidt.
Ó 2008 by the Biophysical Society
0006-3495/08/03/2204/08 $2.00 doi: 10.1529/biophysj.107.111013
Mechanical Properties of Collagen Fibril 2205
was kept ,5°C. The resulting mixture was filtered using a 74-mm filter
properties of nanoscale beams and wires (19 22). This
(collector screen 200 mesh, Bellco Glass, Vineland, NJ). The helical content
method has been applied to silicon beams (19), ZnS nano-
of the collagen suspension after filtration was determined by FTIR (FTS-60,
wires (21), SiO2 nanowires (23), and most recently to elec-
Biorad, Hercules, CA) according to a method described by Friess and Lee
trospun polymer-ceramic composites (24) and individual
(29). After filtration, 1 ml of the collagen dispersion was diluted with 150 ml
amyloid fibrils (25). By use of the same principle, bending
of PBS (pH ź 7.4). Deposition of the collagen fibrils on the quartz glass
substrates was done by incubating the substrates for 10 min in the diluted
of single-walled carbon nanotubes (26) and microtubules
collagen dispersion. Subsequently, the substrates were washed with PBS for
(27) has been performed with contact-mode AFM. In their
10 min and three times with demineralized water for 10 min each and finally
measurements (26,27), the bending moduli (Ebending) related
dried at ambient conditions for at least 24 h. The bending tests of collagen
to the bending stiffness (EbendingI) representing the resistance
fibrils in PBS buffer were carried out after equilibration of the fibrils for 15
of the material on bending were determined (I is the second
min in PBS at room temperature. Longer equilibration times did not lead to
changes in the results of the bending tests.
moment of area of the beam or tube). From the unit-load
Cross-linked collagen fibrils were prepared by mixing 2 ml of the non-
equation, the shear moduli of tested materials were deter-
diluted collagen dispersion with a solution of 1.73 g 1-ethyl-3-(3-dimethyl
mined by bending the materials with different length/diam-
aminopropyl)carbodiimide hydrochloride (EDC) and 0.45 g N-hydroxy-
eter ratios. The determined shear moduli of single-wall
succinimide (NHS) in 215 ml 2-morpholinoethane sulfonic acid (MES)
carbon nanotubes and microtubules are two or three orders of
(0.05 M, pH ź 5.4) for 2 h. The resulting cross-linked fibrils were deposited
on the quartz glass substrates and washed as described above.
magnitude lower than the Young s modulus, which confirms
the mechanical anisotropy of the materials. Adapting the
same technique, mechanical anisotropy in single vimentin
Collagen denaturation temperature and free
intermediate filaments (IFs) was determined (28). This ex-
amino group content
perimental approach offers new insights in separating the
The diluted native collagen fibril dispersion was centrifuged for 15 min at
contribution of the actin filaments, microtubules, and vi-
4500 rpm (Hettich Mikco Rapid/k, Depex, De Bilt, the Netherlands). The
mentin IF networks to the stiffness of the cytoskeleton (28).
solution was removed, and the collagen was washed twice with MilliQ water
To gain more insight into the mechanical behavior of tis-
for 30 min each. Similarly, a diluted cross-linked collagen fibril dispersion
sues, an AFM-based bending technique was developed to
was centrifuged as described above and then washed twice with PBS buffer
study the mechanical behavior of single collagen type I fibrils for 30 min each and four times with MilliQ water for 30 min each. After the
washing steps, both native and cross-linked collagen samples were frozen in
isolated from bovine Achilles tendon. Using a home-built
liquid nitrogen and subsequently freeze-dried for 24 h.
AFM system and a glass substrate with microchannels, fibril
The degree of cross-linking of the collagen samples is related to the in-
bending by cantilever movement in the z-direction was com-
crease of the denaturation (shrinkage) temperature (Td) after cross-linking.
bined with a continuous scanning motion along the fibril. In
The Td values were determined by DSC (DSC 7, Perkin Elmer, Norwalk,
this way, the slope of the force-displacement curve (dF/dz) at CT). Freeze-dried native and cross-linked collagen samples of 3 5 mg were
swollen in 50 ml of PBS (pH ź 7.4) in high-pressure pans overnight. Samples
different positions of the fibril spanning a channel can be ob-
were heated from 20°C to 90°C at a heating rate of 5°C/min. A sample
tained. The bending moduli of tested single collagen fibrils
containing 50 ml of PBS (pH ź 7.4) was used as a reference. The onset of the
were determined by fitting the slope (dF/dz) of multiple indi-
endothermic peak was taken as the Td.
vidual bending experiments to well-established mechanical
The free amino group content of native and cross-linked samples was
models. This method allows a more accurate determination of determined using the 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay.
Collagen samples of 3 5 mg were incubated for 30 min in 1 ml of a 4 wt %
the bending modulus and allowed the calculation of the shear
solution of NaHCO3. To this mixture 1 ml of a freshly prepared solution of
modulus of a collagen fibril for the first time, to our knowl-
TNBS (0.5 wt %) in 4 wt % NaHCO3 was added. The resulting mixture was
edge. Chemical cross-linking is often necessary to improve the
left for 2 h at 40°C. After the addition of HCl (3 ml, 6 M), the temperature was
stability of collagen-based biomaterials. Therefore, the change
raised to 60°C. Degradation of collagen was achieved within 90 min. The
of the mechanical properties on cross-linking the fibrils was resulting solution was diluted with 5.0 ml MilliQ water and cooled to room
temperature. The absorbance at 420 nm was measured using a Varian Cary
investigated.
300 Bio spectrophotometer (Middelburg, the Netherlands). A blank was
prepared by applying the same procedure, except that HCl was added before
the addition of TNBS. The absorbance was correlated to the concentration of
MATERIALS AND METHODS
free amino groups using a calibration curve obtained with glycine. The free
amino group content was expressed as the number of free amino groups per
Quartz glass substrates with parallel microchannels were prepared by reac-
1000 amino acids (n/1000).
tive ion etching using a RIE Elektrotech system (Elektrotech Twin PF 340,
London, UK). The width and depth of the channels were determined by AFM
(home-built instrument) and SEM (LEO Gemini 1550 FEG-SEM, LEO
Micromechanical bending in scanning mode
Elektronenmikroskopie GmbH, Oberkochen, Germany) measurements.
using AFM
Modified triangular silicon nitride cantilevers (coated sharp microlevers
Isolation and deposition of single collagen fibrils
MSCT-AUHW, type F, spring constant k ź 0.5 N/m, Veeco, Cambridge,
Insoluble bovine Achilles tendon collagen type I from Sigma-Aldrich UK) were used in the bending test. The tip on the AFM cantilever was re-
(Steinheim, Germany) was swollen in hydrochloric acid (0.01 M) overnight moved using a focused ion beam (FIB) (FEI, NOVALAB 600 dual-beam
at 0°C. The resulting slurry was homogenized for 10 min at 9500 rpm using a machine). After the cutting, the modified cantilevers were inspected using the
Braun MR 500 HC blender (Braun, Kronberg, Germany). The temperature built-in SEM (30). The spring constant of each tipless cantilever was cali-
Biophysical Journal 94(6) 2204 2211
2206 Yang et al.
brated by pushing on a precalibrated cantilever as described elsewhere (31).
The helical content of the collagen in the fibrillar suspen-
The sensitivity (S) of the AFM system with the cantilever, i.e., the ratio
sion was determined with FTIR and revealed a maximum
between the bending of the cantilever and the deflection, as measured by the
percentage of helicity (29). The single collagen fibrils used
quadrant detector, was derived from a force-indentation curve measured on a
were also characterized by determining their characteristic
glass surface with an identical scan rate and amplitude as used in the bending
denaturation temperature (Td) and number of free amino
experiments.
groups (n/1000). The Td of the native fibrils was 55.0°C and
increased to 74.5°C after cross-linking with the water-soluble
RESULTS AND DISCUSSION
carbodiimide EDC in the presence of N-hydroxysuccinimide
(NHS). The free amino group content decreased from 28 per
Sample preparation and characterization
1000 amino acids to a value of 8, which is in line with pre-
AFM images of the quartz glass substrates (Fig. 1 A) show
viously reported data (32). These results reveal a high degree
that ion etching allows the preparation of a substrate with
of cross-linking.
well-defined microchannels with a width of ;3 mm. The
depth of the channels is ;600 nm, which is sufficient for the
intended bending experiments of collagen fibrils spanning
Micromechanical bending of native and
these channels and supported by the glass rims.
cross-linked collagen type I fibrils
The glass substrates were incubated in a freshly prepared
and highly diluted suspension of collagen fibrils. After Under an optical microscope, collagen fibrils that freely and
washing and drying of the samples, single fibrils perpendic- perpendicularly span multiple channels of the glass substrate
ularly spanning the microchannels were selected and used in were selected for the bending tests. The actual scanning
the scanning-mode mechanical bending tests (vide supra). bending procedure was started after a successful approach of
The characteristic 67-nm D-period of the collagen fibrils the AFM tipless cantilever above the fibril. In scanning mode,
deposited on the glass surface was visualized by AFM im- fibril bending by cantilever movement in the z-direction was
ages both for fibrils at ambient conditions (Fig. 1 B) and in combined with a continuous scanning motion along the fibril.
PBS buffer (Fig. 1 C). Collagen fibrils at least 50 mm in To achieve this, the output signal for the fast scanning di-
length crossed more than 3 channels on the glass substrate rection as used in AFM scanning was used to drive the piezo
(Fig. 1 D). The diameters of all tested fibrils were determined movement in the z-direction while the one for the slow
by high magnification SEM images. scanning direction was used to move the cantilever along the
FIGURE 1 (A) Tapping mode AFM height image of a
glass surface patterned with channels; the full z-range of
the image is 1 mm. (B) Tapping mode AFM height image
of single collagen fibrils on a glass surface at ambient
conditions; the full z-range of the image is 250 nm. (C)
Tapping mode AFM height image of single collagen fibrils
on a glass surface in PBS buffer; the full z-range of the
image is 225 nm. (D) SEM image of a single collagen fibril
spanning multiple channels. The width of the channel is
3.0 6 0.2 mm.
Biophysical Journal 94(6) 2204 2211
Mechanical Properties of Collagen Fibril 2207
fibril (Fig. 2). During the bending tests, the total scanning
distance was chosen to be 4 5 mm, which is slightly larger
than the channel width. A typical piezo movement of 1.5 3.0
mm with a frequency of 1.3 Hz in the z-direction was applied.
In each step, one cycle (approach and retraction) of the
cantilever deflection and piezo movement was recorded.
After every step, the tip was moved one step further along the
fibril. A complete scan consisted of 256 steps with a total
measuring time of 200 s.
Four of the individual piezo movement-deflection curves
obtained from bending the fibril at the middle point of the
channel (a), between the middle and edge of the channel (b),
and at the edge of the channel (c), and indenting the fibril on
FIGURE 3 Four individual piezo movement-versus-deflection curves ob-
the glass surface (d) are presented in Fig. 3. During the first
tained at different positions along the fibril (diameter 240 nm) (a) at themiddle
of the channel, (b) between the middle and edge of the channel, (c) at theedge
part of the approach, there is no deflection, indicating that the
of the channel, (d) on the glass surface. (Inset) Enlargement of the snap-in
cantilever is not interacting with the collagen fibril. As the
points in the same piezo movement-versus-deflection curves. The scale units
cantilever moves closer, a snap-in point can be observed
are 10 nm and 5 nm for horizontal axis and vertical axis, respectively.
(negative deflection). After this point a linear relation be-
tween the piezo movement and deflection of the fibril is
found for all individual bending measurements. The slope of
The slope of each force-displacement curve of the tested
the piezo movement-deflection curve differs from one posi-
fibril was determined by linear fit, and the obtained data are
tion to the next.
presented in Fig. 4. A decrease in the slope (dF/dz) was found
A custom computer program, written in Labview (version
during scanning from the edge up to the middle of the
6.1, National Instruments, Austin, TX) was used to analyze
channel, which clearly proves that the fibril is freely sus-
the data. A force-displacement curve of every 256 bending
pending the microchannel. In all experiments, no difference
measurements was obtained using the following equations:
was found in the force-displacement curves on bending the
same collagen fibril multiple times, which ensures the re-
z ź A D (1)
producibility of the test and confirms that no permanent de-
F ź D 3 k; (2)
formation of the collagen fibrils occurred. It must be noted
in which z is the displacement of the fibril in the z-direction that, in the measurements near the edges of the channel, the
during bending, A is the piezo movement in the z-direction, cantilever can touch the glass surface when the fibril is bent,
and D is the calibrated deflection signal of the cantilever and those data were omitted from analysis.
(nm). F is the force applied to the fibril, and k is the calibrated When a force is applied to the suspended part of the fibril, a
possible displacement in the z-direction at both rims of the
spring constant of the cantilever.
channel has to be taken into account. Because of the strong
FIGURE 2 Schematic representation of the cantilever movement over a
single collagen type I fibril during the scanning-mode bending experiments. FIGURE 4 Slope of the force-versus-displacement curve of a collagen
Each cycle (approach and retraction) of the cantilever movement in the type I fibril (diameter 240 nm) as a function of the scanning position along
z-direction gives a piezo movement-deflection curve. After each cycle, the the channel. The dashed line in the image indicates the middle point of the
cantilever moves one step further along the fibril. In total, 256 steps along channel (channel width is ;3.2 mm). The dF/dz data at the left half and right
the fibril gave 256 piezo movement-deflection curves. half of the channel are fitted to Eq. 3 separately.
Biophysical Journal 94(6) 2204 2211
2208 Yang et al.
surface adhesion properties of collagen to glass (15) and the middle point using Eq. 4, which is derived from Eq. 3 by
fact that each collagen fibril crosses at least three channels substituting x ź l/2
(the length of the fibril is more than 50 mm), it is assumed that
l3 dF
the fibril is firmly attached to the surface at the supporting
Ebending ź 3 : (4)
rims and that the rim behaves as a stiff material, and thus, the
192I dz
displacement can be neglected. Also, it has been reported that
Compared with a single-point bending procedure, the scanning-
slippage of a collagen fibril on the supporting points or
mode bending allows a more precise determination of the
loading points during the bending tests will result in a non-
bending modulus (Young s modulus for isotropic materials
linear force-displacement curve (33). This nonlinearity was
or high length/diameter ratio) because it results from fitting
not observed in our experiments.
multiple individual bending experiments. Furthermore, data
generated from multiple bending experiments of the sus-
pended fibril and fitted to the model of bending a rod reveal
Determining the mechanical properties of native
that no permanent deformation of the collagen fibril occurred
and cross-linked single collagen fibrils
during the bending tests. The relative error (;23%) in the
bending modulus using the applied scanning-mode bending
Deflections of a rod induce both bending and shear defor-
method is derived from the error (SE) of the diameter (;3%),
mation. A bending modulus Ebending as previously defined by
the width of the channel (;3%), the spring constant of the
Kis et al. (27) equals the Young s modulus (E) if the rod is
cantilever (;5%), and the fitting (;3%). The largest contri-
isotropic or the length/diameter ratio fulfills the following
pffiffiffiffiffiffiffiffiffi
bution to the error of the bending modulus results from the
requirement: L=R $ 4 E=G; where G is the shear modulus.
error in the diameter of the fibril (;3%). This leads to a 12%
The Ebending of the suspended fibril can be obtained by fitting
error in the bending modulus. Improvement of the accuracy
the measured slope of the force-displacement curves at all
in the fibril diameter determination is critical for reducing the
positions to Eq. 3 (34),
error further.
The ranges of the bending modulus values that were ob-
dF 3 3 l3 3 Ebending 3 I
ź ; (3)
tained from the scanning-mode bending tests are presented in
dz
ðl xÞ3 3 x3
Table 1. Typically, the bending modulus of a collagen fibril
in which x is the relative position along the fibril (0 # x # l/ with a diameter of 240 nm is ;2.4 GPa (Fig. 4) at ambient
2), l is the width of the channel, I is the moment of inertia conditions. The bending modulus of such a fibril decreased
1
ðI ź pR4Þ; and dF/dz is the slope deduced from the force- with a factor of ;20 to 120 MPa when immersed in PBS
4
displacement curve obtained during bending of the collagen buffer. Introducing cross-links between collagen molecules
fibril. The fibril is considered a rod with a circular cross- by activation of carboxylic acid groups of glutamic or as-
section with radius R. partic acid residues with a carbodiimide, which subsequently
As shown in Fig. 4, the dF/dz data do fit to Eq. 3. The react with amine groups with the formation of amide bonds
standard error in the least-squares fit parameter is 2 6%, and (32) and with hydroxyl groups with the formation of ester
values obtained from the left and right halves of the fibril are bonds (35), did not significantly affect the bending modulus
similar (average difference 3 4%). of the fibril.
Recently we reported on the determination of the Young s For isotropic rods or rods with high length/diameter ratio,
modulus of dry collagen fibrils by single-point bending tests the bending modulus is equal to the Young s modulus and is
(30). The Young s modulus of a fibril crossing a channel in a independent of the rod diameter. Otherwise, the contribution
poly(dimethylsiloxane) substrate was determined close to its of shear in the deflection of the rod can not be ignored. The
TABLE 1 Bending and shear moduli of collagen type I fibrils obtained from scanning bending measurements
Collagen type I fibril Conditions Number of samples Range of diametersz (nm) Bending moduli{ (GPa) Shear modulusż (MPa)
Native Dry 18 187 305 3.9 1.0 33 6 2
Cross-linked* Dry 11 205 303 3.1 1.7 74 6 7
Native PBS buffery 12 280 426 0.17 0.07 2.9 6 0.3
Cross-linked* PBS buffery 13 287 424 0.14 0.06 3.4 6 0.2
*Cross-linking was performed with EDC and NHS in MES buffer.
y
PBS: phosphate-buffered saline, pH ź 7.4.
z
Ranges of diameters of different fibrils used in the mechanical tests. The error in the diameter of individual fibrils is ;3% (SE) calculated from multiple
measurements on the same fibril.
{
Ranges of bending moduli determined from fibrils with different diameters. A 23% relative error is estimated for the value of the bending modulus
determined for individual fibrils.
ż
The error in the shear modulus is the standard error of the weighted least-squares fit parameter.
Biophysical Journal 94(6) 2204 2211
Mechanical Properties of Collagen Fibril 2209
deflection from bending and shear deformation when a force
is applied at the middle of the channel can be written as (34):
z ź zB 1 zS ź Fl3=192EI 1 fsFl=4GA ź Fl3=192EbendingI:
(5)
In Eq. 5, z is the total displacement of the fibril in the
z-direction, zB is the deflection resulting from bending, zS is
the deflection resulting from shearing, E is the Young s
modulus, G is the shear modulus, fs is the form factor of
shear, and A is the cross-sectional area of the rod. For a rod
with a circular cross-sectional area, the form factor of shear
fs equals 10/9 (34).
Equation 5 can be converted into Eq. 6 using A ź pR2,
fs ź 10/9 and I ź pR4/4.
1 1 120 R2
ź 1 3 ð Þ: (6)
Ebending E 9G
l2
From Eq. 6, a diameter-dependent bending modulus is
expected. Such a diameter-dependent bending modulus was
observed before in microtubules (27) and single-wall nano-
tube ropes (26) with relatively weak bonds between the
subunits in the lateral direction. Here, a large number of
fibrils were tested, and we found that the bending modulus
increased with decreasing fibril diameter at both ambient
conditions and in PBS buffer (Fig. 5, A and B).
FIGURE 5 Bending moduli of collagen type I fibrils as a function of
By use of Eq. 6, the shear modulus of the tested collagen
diameter at ambient conditions (A) and in PBS buffer (B). Data points are of
fibrils can be determined from the slope of the linear relation
native collagen fibrils (filled squares) and collagen fibrils cross-linked by
EDC/NHS (open squares). N ź 18 for native collagen (at ambient condi-
between 1/Ebending and (R2/l2). A similar equation has been
tions); N ź 11 for cross-linked collagen (at ambient conditions); N ź 12 for
used by Kis et al. (27) for studying the bending and shear
native collagen (in PBS buffer); and N ź 13 for cross-linked collagen (in
modulus of microtubules. With the linear fit as shown in Fig.
PBS buffer). The relative error in the bending modulus of every individual
6 A, the shear modulus of native single collagen fibrils at
fibril is derived from the errors in fibril diameter (SEM measurements), the
ambient conditions is 33 6 2 MPa. After cross-linking with
length of the channel, and the spring constant of the cantilever.
EDC/NHS, the shear modulus increases to 74 6 7 MPa.
Also, as shown in Fig. 6 B, the shear modulus of the collagen
fibrils placed in PBS buffer can be estimated from the linear resulting from the surface decoration with activated carbox-
plot. The values of the shear moduli are 2.9 6 0.3 MPa and ylic acid groups. In PBS buffer, the surface decoration does
3.4 6 0.2 MPa for native and EDC/NHS cross-linked fibrils, not hamper the displacement of microfibrils with respect to
respectively, which are not statistically different. The dif- each other. It is expected that after cross-linking, the dis-
ferences in the increase of the shear modulus for fibrils at placement of collagen molecules with respect to each other
ambient conditions and when placed in PBS buffer before becomes more difficult. However, the similar shear moduli
and after cross-linking may relate to the different hydration for native and cross-linked collagen fibrils placed in buffer
states of the fibrils. Intermolecular cross-links in collagen indicate that the displacement of microfibrils with respect to
fibrils are mainly present in the telopeptide regions. Cross- each other is probably the main factor influencing the shear
linking using a carbodiimide such as EDC involves the for- modulus of single collagen fibrils. The values of shear moduli
mation of additional amide bonds by reaction of free amine for different collagen fibrils at different conditions are listed
groups (lysine residues) and activated carboxylic acid groups in Table 1. In a previous study (30), the diameter-dependent
(glutamic and aspartic acid) and ester bonds by reaction of bending modulus was not observed because we used chan-
hydroxyl groups (serine, hydroxyproline, and hydroxylysine nels with a larger resulting in a higher length/diameter
pwidth,
ffiffiffiffiffiffiffiffiffi
residues) and activated carboxylic acid groups as well, which ratio (L=R $ 4 E=G); therefore, the Ebending corresponds
results in additional inter- and intramolecular cross-links in more closely with the Young s modulus.
the collagen fibrils. It is not expected that cross-links can be According to current models described in literature
formed between microfibrils because the distance is too long. (2,3,36,37), collagen molecules and microfibrils are arranged
However, displacement of microfibrils with respect to each parallel to the fibril axis. Intermolecular cross-linking for
other at ambient conditions may be hampered by the friction native collagen is believed to occur only via lysine and hy-
Biophysical Journal 94(6) 2204 2211
2210 Yang et al.
different positions across the channel. From the slope of these
curves (dF/dz), the bending modulus of the fibrils could be
determined using an elastic rod model. For single collagen
type I fibrils immersed in buffer, the bending modulus de-
creased by a factor of 20 compared with fibrils at ambient
conditions. Cross-links introduced on reaction with a car-
bodiimide did not change the bending modulus of the fibril.
The dependence of the bending modulus on the collagen
fibril diameter allowed for the first time, to our knowledge, an
estimation of the shear modulus. The calculated shear mo-
dulus indicates that the collagen fibrils are mechanically
anisotropic. For collagen fibrils in PBS buffer, it is shown that
cross-linking through amide bond formation between amine
and carboxylic acid groups and ester bond formation between
hydroxyl and carboxylic acid groups does not affect the shear
modulus of the fibril. These results provide new insight into
the mechanical behavior of collagen-based tissues.
This research was financially supported by the Softlink program of ZonMw.
Project number: 01SL056.
REFERENCES
1. Veis, A. 1997. Collagen fibrillar structure in mineralized and non-
mineralized tissues. Curr. Opin. Solid St. Mater. Sci. 2:370 378.
FIGURE 6 Reciprocal bending modulus (Ebending) as a function of R2/l2 of 2. Piez, K. A., and B. L. Trus. 1981. A new model for packing of type-I
collagen molecules in the native fibril. Biosci. Rep. 1:801 810.
collagen type I fibrils at ambient conditions (A) and in PBS buffer (B). R is
the collagen fibril radius, and l is the suspended length of the fibril over the
3. Orgel, J. P. R. O., T. C. Irving, A. Miller, and T. J. Wess. 2006.
channel. Data points and linear fit are of native collagen type I fibrils (solid Microfibrillar structure of type I collagen in situ. Proc. Natl. Acad. Sci.
USA. 103:9001 9005.
squares; fit, solid line) and collagen type I fibrils cross-linked by EDC/NHS
(open squares; fit, dashed line). N ź 18 for native collagen (at ambient
4. Wess, T. J., A. P. Hammersley, L. Wess, and A. Miller. 1998.
conditions); N ź 11 for cross-linked collagen (at ambient conditions); Molecular packing of type I collagen in tendon. J. Mol. Biol. 275:
255 267.
N ź 12 for native collagen (in PBS buffer); and N ź 13 for cross-linked
collagen (in PBS buffer).
5. Habelitz, S., M. Balooch, S. J. Marshall, G. Balooch, and G. W.
Marshall. 2002. In situ atomic force microscopy of partially deminer-
alized human dentin collagen fibrils. J. Struct. Biol. 138:227 236.
6. Hoffmeister, B. K., S. M. Handley, S. A. Wickline, and J. G. Miller.
droxylysine groups within the telopeptide regions (3). The 1996. Ultrasonic determination of the anisotropy of Young s modulus
of fixed tendon and fixed myocardium. J. Acoust. Soc. Am. 100:3933
interaction between the collagen subunits in the parallel di-
3940.
rection will be different from that in the lateral direction,
7. Kostyuk, O., H. L. Birch, V. Mudera, and R. A. Brown. 2003.
leading to mechanical anisotropy. Previously, microtensile
Structural changes in loaded equine tendons can be monitored by a
tests of single collagen fibrils have been performed in our novel spectroscopic technique. J. Physiol. 554:791 801.
group. The Young s modulus of single collagen fibrils was 8. Fan, Z., P. A. Smith, E. C. Eckstein, and G. F. Harris. 2006.
Mechanical properties of OI type III bone tissue measured by nano-
determined to be 5 6 2 GPa and 0.2 0.5 GPa at ambient
indentation. J. Biomed. Mater. Res. A. 79:71 77.
conditions and in PBS buffer, respectively (17). The shear
9. Elliott, D. M., D. A. Narmoneva, and L. A. Setton. 2002. Direct
modulus of the single collagen fibrils determined from the
measurement of the Poisson s ratio of human patella cartilage in
bending experiments is two orders of magnitude lower than tension. J. Biomech. Eng. 124:223 228.
the Young s modulus, which confirms anisotropy at the 10. Akkus, O. 2005. Elastic deformation of mineralized collagen fibrils: an
equivalent inclusion based composite model. J. Biomech. Eng. 127:
single-collagen-fibril level.
383 390.
To summarize, in this study, the mechanical properties of
11. Natali, A. N., P. G. Pavan, E. L. Carniel, M. E. Lucisano, and G.
insoluble collagen type I fibrils isolated from tendon were
Taglialavoro. 2005. Anisotropic elasto-damage constitutive model for
investigated using scanning-mode bending tests with a home- the biomechanical analysis of tendons. Med. Eng. Phys. 27:209 214.
built AFM. Single fibrils perpendicularly spanning multiple 12. Bischoff, J. E. 2006. Reduced parameter formulation for incorporating
fiber level viscoelasticity into tissue level biomechanical models. Ann.
channels in a glass substrate were subjected to bending tests
Biomed. Eng. 34:1164 1172.
using an AFM cantilever without a tip.
13. Wu, J. Z., and W. Herzog. 2002. Elastic anisotropy of articular
Subjecting a single collagen fibril to the scanning-mode
cartilage is associated with the microstructures of collagen fibers and
bending test afforded multiple force-displacement curves at chondrocytes. J. Biomech. 35:931 942.
Biophysical Journal 94(6) 2204 2211
Mechanical Properties of Collagen Fibril 2211
14. Hellmich, C., J. F. Barthélémy, and L. Dormieux. 2004. Mineral- moduli of single-walled carbon nanotube ropes. Phys. Rev. Lett. 82:
collagen interactions in elasticity of bone ultrastructure-a continuum 944 947.
micromechanics approach. Eur. J. Mech. A/Solid. 23:783 810.
27. Kis, A., S. Kasas, B. Babić, A. J. Kulik, W. Benoît, G. A. D. Briggs, C.
15. Graham, J. S., A. N. Vomund, C. L. Phillips, and M. Grandbois. 2004. Schönenberger, S. Catsicas, and L. Forró. 2002. Nanomechanics of
Structural changes in human type I collagen fibrils investigated by microtubules. Phys. Rev. Lett. 89:248101-1 248101-4.
force spectroscopy. Exp. Cell Res. 299:335 342.
28. Guzmán, C., S. Jeney, L. Kreplak, S. Kasas, A. J. Kulik, U. Aebi, and
16. Eppell, S. J., B. N. Smith, H. Kahn, and R. Ballarini. 2006. Nano L. Forró. 2006. Exploring the mechanical properties of single vimentin
measurements with micro-devices: mechanical properties of hydrated
intermediate filaments by atomic force microscopy. J. Mol. Biol. 360:
collagen fibrils. J. R. Soc. Interface. 3:117 121.
623 630.
17. van der Rijt, J. A. J., K. O. van der Werf, M. L. Bennink, P. J. Dijkstra,
29. Friess, W., and G. Lee. 1996. Basic thermoanalytical studies of
and J. Feijen. 2006. Micromechanical testing of individual collagen
insoluble collagen matrices. Biomaterials. 17:2289 2294.
fibrils. Macromol. Biosci. 6:697 702.
30. Yang, L., K. O. van der Werf, H. F. J. M. Koopman, V. Subramaniam,
18. Wenger, M. P. E., L. Bozec, M. A. Horton, and P. Mesquida. 2007.
M. L. Bennink, P. J. Dijkstra, and J. Feijen. 2007. Micromechanical
Mechanical properties of collagen fibrils. Biophys. J. 93:1255 1263.
bending of single collagen fibrils using atomic force microscopy.
J. Biomed. Mater. Res. A. 82:160 168.
19. Namazu, T., Y. Isono, and T. Tanaka. 2000. Evaluation of size effect
on mechanical properties of single crystal silicon by nanoscale bending
31. Torii, A., M. Sasaki, K. Hane, and S. Okuma. 1996. A method for
test using AFM. J. Microelectromech. Syst. 9:450 459.
determining the spring constant of cantilevers for atomic force micros-
copy. Meas. Sci. Technol. 7:179 184.
20. Ni, H., and X. Li. 2006. Young s modulus of ZnO nanobelts measured
using atomic force microscopy and nanoindentation techniques. Nano-
32. Olde Damink, L. H. H., P. J. Dijkstra, M. J. A. van Luyn, P. B. van
technology. 17:3591 3597.
Wachem, P. Nieuwenhuis, and J. Feijen. 1996. Cross-linking of dermal
21. Xiong, Q., N. Duarte, S. Tadigadapa, and P. C. Eklund. 2006. Force- sheep collagen using a water-soluble carbodiimide. Biomaterials. 17:
765 773.
deflection spectroscopy: a new method to determine the Young s
modulus of nanofilaments. Nano Lett. 6:1904 1909.
33. Ljungcrantz, H., L. Hultman, J. Sundgren, S. Johansson, N. Kristensen,
and J. Schweitz. 1993. Residual stresses and fracture properties of mag-
22. Jeng, Y. R., and P. C. Tsai. 2005. Molecular-dynamics studies of
bending mechanical properties of empty and C60-filled carbon nano- netron sputtered Ti films on Si. microelements. J. Vac. Sci. Technol. A.
11:543 553.
tubes under nanoindentation. J. Chem. Phys. 122:224713.
34. Gere, J. M., and S. P. Timoshenko. 1991. Mechanics of materials, 3rd
23. Ni, H., X. Li, and H. Gao. 2006. Elastic modulus of amorphous SiO2
edition. Chapman & Hall, London.
nanowires. Appl. Phys. Lett. 88:043108.
35. Everaerts, F., M. Torrianni, M. Hendriks, and J. Feijen. 2007. Quan-
24. Lee, S. H., C. Tekmen, and W. M. Sigmund. 2005. Three-point
tification of carboxyl groups in carbodiimide cross-linked collagen
bending of electrospun TiO2 nanofibers. Mater. Sci. Eng. A/Struct. 398:
77 81. sponges. J. Biomed. Mater. Res. A. 83:1176 1183.
25. Smith, J. F., T. P. J. Knowles, C. M. Dobson, C. E. MacPhee, and M. E. 36. Buehler, M. J. 2006. Nature designs tough collagen: Explaining the
Welland. 2006. Characterization of the nanoscale properties of individual nanostructure of collagen fibrils. Proc. Natl. Acad. Sci. USA. 103:
amyloid fibrils. Proc. Natl. Acad. Sci. USA. 103:15806 15811. 12285 12290.
26. Salvetat, J. P., G. A. D. Briggs, J. M. Bonard, R. R. Bacsa, A. J. Kulik, 37. Prockop, D. J., and A. Fertala. 1998. The collagen fibril: the almost
T. Stöckli, N. A. Burnham, and L. Forró. 1999. Elastic and shear crystalline structure. J. Struct. Biol. 122:111 118.
Biophysical Journal 94(6) 2204 2211
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