Influence of Fibrinogen Concentration On Young's Modulus in Fibrin Gels

Influence of fibrinogen
concentration on the Youngs

modulus in fibrin gels
BMTE02.44
Julienne Brouwers
Supervisors: Drs. M. van Lieshout, Dr. Ir. G. Peters

Eindhoven, November 2002
Influence of fibrinogen concentration in fibrin gels on Youngs modulus
Abstract
For tissue engineering a heart valve, the idea has risen to combine the advantages of
polycaprolactone and fibrin by using the first as a mechanical backbone and the latter as a
cell-delivery substance surrounding the backbone. The mechanical properties of fibrin should
still be high enough to be used as part of a scaffold for a heart valve. Therefore, a
reproducible protocol has been developed to determine the Youngs modulus using a tensile
testing machine. Also, the influence of fibrinogen concentration in fibrin gels on the Youngs
modulus has been determined, by using the developed protocol. The protocol consisted of
making gels, which originated from mixing a fibrinogen solution with a thrombin solution.
This was done for fibrinogen solutions with a concentration of 4, 8, 10, 14, 18 and 30 mg/ml
and a thrombin solution of 5 IU/ml. Then the fibrin gels were placed in the tensile testing
machine with dimensions 5 mm in width and 30 mm in length. The results showed a linear
increasing Youngs modulus with increasing strain for all samples. The correlation between
the Youngs modulus and the concentration was linearly increasing until a concentration of
about 14 mg/ml showing a maximum initial Youngs modulus of 9995 Pa (+-185.5) and a
maximum Youngs modulus at +-20 % strain of 29600 Pa (+-932.8). The reproducibility was
higher at high fibrinogen concentrations than at lower ones. The developed protocol is
especially suited for the testing of fibrin gels with a high fibrinogen concentration.
Department of Biomedical Engineering 2
Table of contents
Abstract ---------------------------------------------------------------------------------------- 2
1. Introduction -------------------------------------------------------------------------------- 4
1.1 Fibrinogen ------------------------------------------------------------------------------ 5
1.1.1 Structure---------------------------------------------------------------------------- 5
1.1.2 Formation of fibrin network ----------------------------------------------------- 6
1.2 Mechanics------------------------------------------------------------------------------- 6
1.2.1 Neo-Hookean ---------------------------------------------------------------------- 7
1.2.1.1 Uniaxial extension -------------------------------------------------------------- 7
1.2.1.2 Planar extension ---------------------------------------------------------------- 9
2. Materials and Methods------------------------------------------------------------------ 10
2.1 Fibrin gel preparation ---------------------------------------------------------------- 11
2.2 Tensile testing ------------------------------------------------------------------------- 11
2.3 Data processing ----------------------------------------------------------------------- 12
2.4 SEM preparation ---------------------------------------------------------------------- 12
2.5 Rheology testing ----------------------------------------------------------------------- 12
3. Results -------------------------------------------------------------------------------------- 14
3.1 Uniaxial extension -------------------------------------------------------------------- 14
3.2 Planar extension ---------------------------------------------------------------------- 17
3.3 SEM ------------------------------------------------------------------------------------- 18
3.4 Rheometer------------------------------------------------------------------------------ 18
Conclusions----------------------------------------------------------------------------------- 19
Discussion------------------------------------------------------------------------------------- 20
Acknowledgements-------------------------------------------------------------------------- 22
References: ----------------------------------------------------------------------------------- 22
Appendix A----------------------------------------------------------------------------------- 23
Appendix B ----------------------------------------------------------------------------------- 24
Appendix C----------------------------------------------------------------------------------- 26
1. Introduction
Tissue engineering has emerged as a rapidly expanding approach to address the organ
shortage problem. It is an interdisciplinary field that applies the principles and methods of
engineering and the life sciences toward the development of biological substitutes that can
restore, maintain, or improve tissue function. Much progress has been made in the tissue
engineering of structures relevant to cardiothoracic surgery, including heart valves, blood
vessels and myocardium [1]. Usually, isolated cells are used to engineer new tissues. The
cells are seeded on a three dimensional scaffold followed by in vitro culturing. The scaffolds
serve as physical supports and templates for cell attachment and tissue development [2].
The requirements for an ideal scaffold are the easy handling and moulding of complex 3-D
structures like valve conduits or vessels with complex side branches. The material should not
be toxic or have any immunologic side effects. The diffusion barrier of the scaffold has to be
as low as possible to guarantee an optimal nutrition in thicker tissues. The mechanical as well
as the chemical properties (e.g. the integration of growth factors) should be modifiable. The
control of the degradation is important to adapt the structural support of the scaffold to the
tissue development. Many substances are used in the production of scaffolds, e.g. synthetic
polymers, natural polymers or biological scaffolds [2].
Fibrin is a biodegradable polymer and the major component of blood clots. It is formed by
polymerisation of fibrinogen monomers catalysed by thrombin. In comparison to other
materials used for tissue engineering purposes, fibrin gel combines some important
advantages. At first, the degradation rate is controllable and can be adapted to the tissue
development. Beyond it, fibrin gel can be produced from patients blood and therefore
immunogenic reactions are improbable [2]. Inhomogeneous distribution of cells in a scaffold
is often a problem. In contrast, when cells are incorporated before gelling, the distribution in
the fibrin gel is homogeneous from the beginning, caused by the fast immobilization of the
cells during the polymerisation of the gel/cell suspension within a few minutes. Structures
with a layer thickness up to 3 mm can be produced without the problem of local cell necrosis
caused by diminished diffusion [2].
A major disadvantage of fibrin is the mechanical stiffness, which seems to be low (table 1.1).
Table 1.1 Details of experiments with fibrin gels.
Reference Quantity
Fibrinogen
Value
concentration range
(mg/ml)
(kPa)
[3]
Youngs
0.5-3
0.93-6.49
modulus
[4]
Storage shear 0-6
0-0.15
modulus
[5]
Youngs
30-70
6.7-23.9
modulus at
10% strain
[6]
Tensile
25-75
2-fold
Strength
increase
[7]
Youngs
modulus
16-28
12
[8]
Tensile
Strength
20-60
5-fold
increase
Correlation
Experiment
Unclear
Uniaxial extension
Unclear
Rheometer
Unclear
Uniaxial extension
Rate=25 mm/min
Directly
proportional to
concentration
of fibrinogen
Not influenced
by fibrinogen
concentration
Unclear
Tensile testing
Tensile testing
Rate= 30 mm/min
Unclear
The influence of the fibrinogen concentration in fibrin on the mechanical properties has been
investigated but researchers have produced varying values for this influence and also for the
Youngs modulus; unfortunately, often the testing protocol is unclear. Moreover, the testing
protocols, if known, are very different.
Other materials have been considered, like polycaprolactone. This material is well suited to be
knitted as a scaffold, which seems to be more mechanically compatible than fibrin. The idea
has risen to combine the advantages of polycaprolactone and fibrin by using the first as a
mechanical backbone and the latter as a cell-delivery substance surrounding the backbone.
The mechanical properties of fibrin should still be high enough to be used as part of a scaffold
for a heart valve. Different combinations of factors leading to the optimal mechanical
properties have to be determined. Therefore, a reproducible mechanical test should be
developed. The preceding has led to the following two questions:
What is the optimal protocol for a reproducible mechanical test on fibrin gel?
What is the influence of the concentration of fibrinogen in the fibrin gel on the Youngs
modulus?
1.1 Fibrinogen
1.1.1 Structure
Following vascular injury, fibrin comprises the major protein component of the hemostatic
plug and fibrinogen is the target protein of the coagulation cascade. It can also be used as
fibrin glue during surgery. Fibrinogen is a 340-kDa soluble plasma protein consisting of
three pairs of disulfide-bonded -, - and -chains arranged as depicted in figure 1.1. The E
and D regions in each half-molecule are delineated by a pair of disulfide rings, which link
chains to , to and to [9].
Figure 1.1 Schematic structure of fibrinogen. Fibrinopeptides A and B are shown as black boxes on the
- and -chains, respectively. The disulfide bonds joining the chains are shown as lines [9].
1.1.2 Formation of fibrin network

Several thrombin-sensitive bonds have been identified and are shown as small arrows in
figure 1.1. Thrombin converts fibrinogen to fibrin by catalysing the proteolytic removal of
both fibrinopeptide A and fibrinopeptide B. These cleavages unmask two polymerisation sites
in the E region, forming soluble fibrin, to which one D region from each of two fibrin(ogen)
molecules can bind. The fibrin spontaneously polymerises to form double-stranded
protofibrils, with the fibrin monomers within each strand end-to-end and the fibrin across
strands arranged in a half-staggered overlap, as depicted by the combination of strands a and b
in figure 1.2 [9].
Figure 1.2 The formation of fibrin network [9].
The protofibrils may further associate laterally to produce fibres, which themselves may
associate to form fibre bundles. As also indicated in figure 1.2, the -chains of adjacent D
regions within a strand of a protofibril are covalently cross-linked by isopeptide bonds, the
formation of which is catalysed by factor XIIIa. Thus, in cross-linked fibrin, the individual
strands consist of polymers of covalently linked fibrin molecules [9].
Covalent cross-linking within fibrin networks produces dramatic effects on rheological
properties. Ligated clots exhibit mechanical stiffnesses up to five times greater than their
unligated counterparts [4].
1.2 Mechanics
Fibrin gel is a visco-elastic material, which means that it exhibits both viscous and elastic
properties. A common approach to modelling visco-elastic behaviour is by means of
combining elements representing ideal elastic behaviour and ideal viscous behaviour. The
usual method of doing this is using a spring and dashpot analogy. The spring represents ideal
elastic properties and the dashpot, ideal viscous properties. The simplest combinations are to
put the two elements in series and in parallel. This leads to the so-called Maxwell element and
the Kelvin or Voigt element. These are illustrated in figure 1.3 [10]. Different combinations
of several Maxwell and Kelvin-Voigt elements can be used to model visco-elastic materials.
Maxwell Element
Kelvin-Voigt Element
Fig 1.3 Spring and Dashpot models for visco-elasticity [10].

If a strain is applied to a visco-elastic material there is an instantaneous response (elastic) and

a delayed response (viscous). A purely elastic response is in phase with the strain and a purely
viscous response is out of phase with the strain. The relationship between the applied strain
and the stress response is expressed for a visco-elastic material by defining two shear moduli,
G' and G". These are known as the storage modulus and the loss modulus respectively. The
storage modulus describes the component of the stress that is in phase with the strain and
indicates the stored energy and the loss modulus describes the component that is out of phase
with the strain and indicates the dissipated energy [10].
The shear modulus is defined as
G = G '+iG ' '
and
G = (G ' 2 + G ' ' 2 )
(1)
1.2.1 Neo-Hookean
Another way to describe the dynamic behaviour of fibrin is by using the neo-Hookean model
[11]. The Neo-Hookean constitutive equation is given by:
T = pI + GE
(2)
where T is the stress, p is the hydrostatic pressure, G is the elastic modulus in shear and E the
strain tensor.
= BI
(3)
B = F * FT
(4)
where F is the deformation gradient tensor.

1.2.1.1 Uniaxial extension
For uniaxial extension with length of the sample>>width of the sample, B becomes:
0
0 1
0
0 12
0
0
1
B = F * FT = 0 1 1/ 2
0 . 0 1 1 / 2
0 = 0 1 1 0
0
11 / 2 0
11 / 2 0
0
0
0 11
(5)
where is the ratio of deformed to undeformed length.

The stress components are calculated by substituting the components of B into eq. 2
T11 = p + G12
T22 = T33 = p +
(6)
G
1
(7)
The deformation is achieved by applying a force f in x1 direction, at the ends of the sample.
The true stress acting on the ends is divided by the area of the deformed sample x2x3=a1,
so that
f1
= T11 = p + G 12
a1
(8)
Because no tractions act on the sides of the block, T22= T33 = 0 and p=G/1. Substituting this
expression in eq. 8 gives
1
f
= T11 = G ( 12 )
a1
1
(9)
For an incompressible material
volume = a1x1 = a1* x1*
or
a1 =
a1*
1
where a1* and x1* are the original area and length.
Eq. 9 can also be expressed in terms of the strain . Recalling that =1+ and substituting,
yields
3 + 3 2 + 3
)
T11 = G (
1+
(10)
In the limit of small strain
T11 = 3G
for
<< 1
(11)
which is in the linear region of figure 1.4.
Figure 1.4. Tensile and compressive stress versus extension ratio for a rubber sample [11].
The Youngs modulus can be defined as
T11 T22
0
E = lim
(12)
which gives
E = 3G
(13)
Thus, the Youngs modulus is three times the modulus in shear, a well known result for
incompressible, isotropic materials.
1.2.1.2 Planar extension

For planar extension with width of sample >> length of sample >> thickness of sample, the
width will change little compared to the length and thickness. B becomes:
1
T
B = F*F = 0
0 1
0 . 0
1
0
0
1
0
1

0 12
0=0
1
0
0
1
0
1
0
0
1
0
12
0
1
(14)
The stress components are calculated by substituting the components of B into eq. 2
T11 = p + G12
T22 = p + G
G
T33 = p + 2
1
(15)
Since there are no forces acting on the x3-surface, T33=0, p=G/21. Thus
T11 = G ( 12
1
)
12
(16)
For small strains it can be deducted that the planar tensile modulus is four times that in
shear
E = 4G
(17)
2. Materials and Methods

Fibrinogen type 1-S from bovine plasma (Sigma) and thrombin from bovine plasma (Sigma)
were both dissolved in medium, which consisted of 90% Hams F-12 with L-Glutamin
(Biowhittaker), 9% fetal bovine serum (Biochrom ag) and 0.9% penicillin/streptomycin
10.000 u/10.000 g/ml (Biochrom ag). A fresh fibrinogen solution was made for every
experiment, but the thrombin solution was used for about 7 days. In order to fill the mould
with fibrinogen and thrombin a double-seringe (two-component fibrin sealant) was used
called Geratzesatz/Kit-5.0 ml (Baxter) (figure 2.1).
Figure 2.1 The two-component fibrin sealant.
It mixes the two components completely right before they enter the mould, so the fibrinogen
starts polymerising once in the mould. All moulds were 4 cm in length, 1 cm in width and 0.5
cm in depth (figure 2.2).
Figure 2.2 The moulds used for the production of fibrin gels.
A Zwick Z010 tensile testing machine with a 20 N-sensor in series with a 10 kN-sensor was
used to do the tensile testing. The clamps used were 1 cm in width. As mentioned in the
section mechanics, pieces of fibrin with length >> width (uniaxial extension) could be used or
pieces of fibrin with width >> length (planar extension). At first, fibrin gels with width >>
length were used to do tensile testing. Then it was decided to apply uniaxial extension with
pieces of fibrin of 5 mm in width and with length >> width.
It was hypothesized that during the experiments the concentration of thrombin in the fibrin
gels was high enough to establish complete polymerisation of the fibrinogen. However, the
amount of thrombin was doubled twice to make sure the concentration was high enough for
complete polymerisation. SEM pictures were taken to get a view of the structure of different
fibrin gels. A few rheology tests were performed to compare the tensile testing results with
rheology results.
2.1 Fibrin gel preparation

The fibrinogen and thrombin needed were weighed. Fibrinogen was dissolved in medium,
often using a stirring bar. Thrombin was dissolved in medium. The double-seringe was filled
with fibrinogen and thrombin solutions. Then the needle was placed on the seringes and the
moulds were filled while the solutions mixed. Then the moulds were placed in the incubator
for 1 hour at 370, which is the optimum temperature for thrombin. Carefully, the fibrin gels
were taken out of the moulds. The pieces for the planar extension were sectioned tranversely
and the pieces for the uniaxial extension were sectioned longitudinally using a scalpel. Each
part of the fibrin gels was weighed to determine its dimensions and put in a petri dish with
medium to prevent from dehydration.
2.2 Tensile testing

The clamps were placed exactly parallel. The fibrin gel was measured in length and then hung
in the upper clamp of the tensile testing machine. When it was hanging freely, straight down,
the force was nulled to rule out the stress created by its own weight. Then the fibrin gel was
let down and tightened in the bottom clamp leaving a distance of approximately 10 mm
between the clamps for the planar extension and 30 mm for the uniaxial extension. Because
often, when tightened, the samples enforced a negative force about -0.003 N, a pre-load of
0.005 N was first applied at a speed of 30 mm/min. In this way, the samples were all stretched
at the same initial load. Then the sample was stretched to a strain of 25% at a speed of 30
mm/min (earlier tests had shown that the strain at break was over 100 % (appendix A)). The
clamps returned back to their original position. The fibrin gel was taken out and placed back
again in the same position as the first time. The sample was tested again to collect more
results (earlier tests had shown that at 25 % strain no plastic deformation had taken place
(Appendix A)). For the planar extension the distance between the clamps was now 5 mm, for
the uniaxial extension it was again 30 mm. Again the force was nulled while the sample was
hanging freely. This was often necessary, because water was pressed out making the weight
less and thus the force smaller. The protocol was the same for all the different concentrations
of fibrinogen (table 2.1). The experiments with short samples were only performed once with
several fibrin gels. Of all experiments with long samples the concentration 30 mg/ml was
tested once, the concentrations 4, 8 and 14 mg/ml were tested twice and the concentration 18
mg/ml was tested three times. Moreover, the concentrations 14 and 18 mg/ml were also tested
once with a doubled amount of thrombin. During every experiment several fibrin gels were
tested.
Table 2.1 Several concentrations of fibrinogen and thrombin, which have been tested.
Concentration
Concentration
Length of clamp sample
fibrinogen (mg/ml)
thrombin (IU/ml)
(mm)
10
5
10/ 5
14
5
10/ 5
4
5
30
8
5
30
14
5
30
14
10
30
18
5
30
18
10
30
30
5
30
Because most concentrations were tested on more than one date, not only the reproducibility
between the fibrin gels of the different moulds could be determined, but also the
reproducibility between different testing dates.
2.3 Data processing

The computer program which switched on the tensile testing machine was programmed to
produce *.Tra files which contained a length of the sample column and a force column.
The area of the sample was calculated by dividing the volume by the length. Because the
fibrin gels almost entirely consist of medium, the density was assumed to be 1g/ml.
The strain was determined using the length of the sample at the time of the pre-load as initial
length. This means that all the samples started at the same force. The advantage is that the
initial length doesnt differ because of different initial forces caused by tightening of the
clamps. Of course, the initial stress wasnt exactly the same for all fibrin gels, because of
different areas, but this was considered negligible.
The tensile testing machine was not developed for materials with a low Youngs modulus like
fibrin. Probably because the 20-N sensor was measuring forces of not even 1 % of its
maximum, the force-strain curves showed irregularities instead of being smooth. Therefore,
the strains and forces were averaged. For data-files containing about 2500 numbers 9 points
before and after each number were summed, all weighed equally, and divided by 19 to
smooth the curve. For data-files containing less numbers, the averaging was done using
approximately the same ratio of 2500/9 (appendix B).
The stress was determined by using eq. 9. The Youngs modulus was determined by taking
the derivative of the stress against 2-1/ for uniaxial extension times three (eq. 11) and by
taking the derivative of stress against 2-1/2 times four for planar extension (eq. 17). For a
Neo-Hookean material the curve should be at least initially flat, but this wasnt the case. It
was assumed that the first part of the curves was linear. Data of the four curves of one fibrin
gel (recall that every sample was sectioned longitudinally and both pieces were tested twice)
were taken together and a linear line was fitted through all data up to 2-1/ =0.25 using
Matlab-tools-datafitting. The first 25 data were left out, because often in the beginning there
were strong irregularities. For data-files containing less numbers, the amount of data left out
using approximately the same ratio of 2500/25. The initial Youngs modulus was defined as
the value of the curve at the x-axis intercept (appendix C).
Then all data of one sample were plotted up to 2-1/ =0.7 and another curve was fitted. A
4th degree polynomial was experimentally determined to be the best fit. Although the
relationship between G and E is only valid for small strains, the Youngs modulus at a strain
of 20 % was nevertheless determined, because this level of strain corresponds with the strains
occurring in a human aortic valve.
2.4 SEM preparation

Fixated or freeze-dried samples were used to make SEM pictures (SEM Philips XL30
Scanning Electron Microscopes). The samples with the highest and the lowest concentration
in this study were used to see if there was any difference in structure. All samples were used
after tensile testing was done.
Two pieces of fibrin of 30 mg/ml were placed in water for an hour and then placed in the
freezer of 800 C together with two pieces of fibrin of 30 mg/ml that came directly out of the
medium. Two pieces of fibrin of 4 mg/ml and two pieces of fibrin of 8 mg/ml that came
directly out of the medium were also placed in the freezer of 800 C. All fibrin pieces were
freeze-dried and gold-sputtered (Labconco FreeZone freeze dry system). Then SEM pictures
were taken. Both samples kept in water and medium were used to rule out the possibility that
salts would reduce the view when SEM pictures were taken.
2.5 Rheology testing

A cylindrical sample of fibrin with concentration 18 mg/ml was prepared according to the
protocol described above. It was placed in the rheometer (Rheometric Scientific). It was
compressed to about 10% and a frequency sweep test was performed. Earlier research found a
strain of 0.5 % to be the optimum strain to measure at. Other strains were applied as well. To
obtain more results the experiment was done with another sample. The rheometer produced
values for the dynamic modulus, which were multiplied by three. These tests were performed
to check whether they would give the same value for the Youngs modulus as the tensile
testing did.
3. Results
3.1 Uniaxial extension
Figure 3.1 shows the non-averaged force against strain for one sample with a fibrinogen
concentration of 18 mg/ml. The two sectioned pieces of a fibrin gel were always slightly
different in area which can be seen in the figure by the two different levels of forces. Also, the
irregularities of the curve are depicted. There was no decline in strength between the first and
the second measurement on one piece of fibrin. Differences in value occurred simply because
of a new measurement. Forces could be slightly higher as well as lower during the second
measurement.
force-strain curves
0.14
0.12
0.1
force (N)
0.08
0.06
0.04
0.02
sample
sample
sample
sample
0
-0.02
0.05
0.1
0.15
A
A
B
B
test
test
test
test
1
2
1
2
0.2
0.25
strain
Figure 3.1 Force-strain curves for all four pieces of one sample with a fibrinogen concentration of 18
mg/ml.
Figure 3.2 shows the stress against 2-1/ for the same sample. The irregularities have
become less by the averaging of the forces and strains, but are still apparent.
stress -strain curves
7000
6000
stress (N/m )
5000
4000
3000
2000
1000
sample
sample
sample
sample
0
-1000
0.1
0.2
0.3
0.4
0.5
0.6
A
A
B
B
test
test
test
test
0.7
1
2
1
2
0.8
alfa - 1/alfa
Figure 3.2 Stress-strain curves for all four pieces of one sample with a fibrinogen concentration of 18
mg/ml.
Therefore, the derivatives of these curves, which have been taken to determine the Youngs
modulus, show an enormous spreading (figure 3.3).
Young's modulus
x 10
Youngs' modulus (Pa)
0.5
0
-0.5
-1
-1.5
-2
sample
sample
sample
sample
-2.5
-3
0.1
0.2
0.3
0.4
0.5
0.6
A
A
B
B
test
test
test
test
1
2
1
2
0.7
0.8
alfa - 1/alfa
Figure 3.3 The Youngs modulus curves for all four pieces of one sample with a fibrinogen
concentration of 18 mg/ml.
Figure 3.4 shows the linearly fitted line through all data of this sample up to 2-1/ =0.25.
5
x 10
Young's modulus
data 1
linear
y = 4.134e+004*x + 9574
4
3
2
1
0
Y o u n g 's
m o d u lu s
(P a )
-1
-2
0.05
0.1
0.15
2
alfa - 1/alfa
0.2
0.25
Figure 3.4 Linear fit through all data up to 2-1/ =0.25.
The initial Youngs modulus here is 9.574 kPa.

Figure 3.5 shows all data of this sample up to 2-1/ =0.7 and the fitted 4Th degree
polynomial. The Youngs modulus at 2-1/ =0.6 (~20 % strain) is 29.76 kPa.
x 10
Young's modulus
Young's modulus (Pa)
6
4
2
0
-2
-4
-6
-8
0.1
0.2
0.3
0.4
0.5
0.6
0.7
alfa - 1/alfa
Figure 3.5 Fitted polynomial through all data of this sample up to 2-1/ =0.7.
For all uniaxial extension tests these four above shown figures were similar.
Table 3.1 shows the calculated averaged Youngs moduli. During every experiment several
fibrin gels were tested. First the average value within one experiment was taken for every
experiment of the same concentration. Then the average of these values was taken. The
standard deviation was determined by averaging the standard deviation of all experiments
with the same concentration. The experiments with doubled thrombin concentration did not
show higher Youngs moduli. Therefore, the results have also been used to calculate the
average modulus and the standard deviation.
Table 3.1 Calculated Youngs moduli for uniaxial extension. Upper standard deviation is the averaged
standard deviation of all experiments with the same concentration. The lower is the standard deviation
of the averaged values of all experiments with the same concentration and thus a measure for the
reproducibility between experiments.
Fibrinogen
Number of
Initial Youngs modulus
Youngs modulus at 2-1/
concentration
experiments
(Pa) (mean +-SD)
=0.6 (Pa) (mean +-SD)
(mg/ml)
4
2
4722 +- 881.6 (18.7%)
10480 +- 2320 (22.1%)
+- 690.8 (14.6 %)
+- 1472 (14.0 %
8
2
5898 +- 889.8 (15.1%)
15060 +- 1041 (6.9%)
+- 276.5 (4.7 %)
+- 438.4 (2.9 %)
14
3
9584 +- 417.4 (4.4 %)
26720 +- 1399 (5.2 %)
+- 2026 (21.1 %)
n.d.
18
4
9809 +- 501.8 (5.1%)
29600 +- 932.8 (3.2%)
+- 495.8 (5.1 %)
+- 63.64 (0.2 %)
30
1
9995 +- 185.5 (1.9%)
28550 +- 1184 (4.2%)
n.d.
n.d.
Figure 3.6 and figure 3.7 depict the upper standard deviation of table 3.1.
Initial Young's modulus

12000
10000
8000
6000
4000
2000
Young's modulus and standard deviation
0
0
10
15
20
25
30
35
concentration fibrinogen (mg/ml)
Figure 3.6 Initial averaged Youngs modulus and standard deviation
Young's modulus at alfa^2 - 1/alfa =0.6
35000
30000
25000
20000
15000
10000
5000
Young's modulus and standard deviation

0
0
10
15
20
25
30
35
Concentration fibrinogen (mg/ml)
Figure 3.7 Averaged Youngs moduli and standard deviation at at 2-1/ =0.6.
3.2 Planar extension

The planar extension experiments using fibrin samples with width >> length resulted in an
enormous spreading and a much lower Youngs modulus than obtained using the other tensile
testing results as can be seen in table 3.2.
Table 3.2 Calculated Youngs moduli for planar extension.
Concentration
Initial Youngs modulus (Pa)
(mean +-SD)
10
3101 +- 600.2 (19.4 %)
14
4604 +- 706.9 (15.4 %)
Youngs modulus at 2-1/

=0.6 (Pa) (mean +-SD)
6421 +- 1188 (18.5%)
14710 +- 433.1 ( 3.0%)
3.3 SEM
The pictures of all samples taken by the SEM showed areas with a different kind of structure,
which would suggest that the fibrin gels were inhomogeneous (figure 3.8). There was no
difference in structure seen between the samples of different concentrations. Also, some
abnormalities were found on the surface as depicted in figure 3.9. In a lot of regions the
structure shown in figure 3.10 was seen.
Figure 3.8 SEM picture (60x) of a fibrin sample with a fibrinogen concentration of 4 mg/ml.
3.4 Rheometer
Both samples had a Youngs modulus of about 3 kPa, with a range from 2.1 to 3.6 kPa.
Conclusions
An optimal protocol for reproducing mechanical testing on fibrin gel has been set up and is
described above. The reproducibility within an experiment and between several experiments
is good. The correlation between the fibrinogen concentration in fibrin gels and the Youngs
modulus is linear for concentrations between 4 and about 14 mg/ml showing a maximum
initial Youngs modulus of 9995 Pa and a maximum Youngs modulus at +-20 % strain of
29600 Pa. Above that, changing the fibrinogen concentration has no influence on the Youngs
modulus. The reproducibility within an experiment is higher at high fibrinogen concentrations
than at all of the lower ones. This applies for the initial Youngs modulus and the modulus at
2-1/ =0.6.
Discussion
Initially, fibrin gels with width >> length were used to do tensile testing. Unfortunately, it
appeared that gels of 1 cm width (recall that the clamps were also 1 cm in width) showed to
bulge out of the clamps once tightened and this affects the measurement by causing a lower
Youngs modulus and a higher standard deviation. Because either fibrin gels with length >>
width or with width >> length had to be used to comply with respectively uniaxial extension
or planar extension it was decided to use pieces of fibrin of 5 mm in width and with length >>
width.
Because it would have taken too long to wait for new moulds with a width of 5 mm it was
decided to section the fibrins longitudinally.
There are several causes for the deviations that are seen. When the fibrins were taken out of
the moulds sometimes they stuck to the mould, especially the fibrins with low concentration
of fibrinogen. Consequently, there were tears or variations in width. Because by dividing the
weight by the length, the averaged area was determined. The samples which had been
damaged had in fact a lower true area, because the smallest area determines the amount of
strain a certain force causes. Also, sometimes the fibrin gels had a slanting side, which also
causes a deviated area.
When sectioning the fibrin gels it was difficult to section them exactly straight. More
difficulties occurred with fibrin gels with a low fibrinogen concentration than with the ones
with a high concentration, because of their weakness. This variance in width influenced the
Youngs modulus slightly.
Sometimes it was not possible to place the fibrin pieces exactly straight in the clamps,
because for example they were a bit twisted. This probably caused shear stresses, which
lowered the calculated Youngs modulus. When tightening the clamps, the fibrin present in
between the clamps was pushed out and caused a negative force. A pre-load was applied to
try to compensate for this but the negative force is not completely caused by compression so
this pre-load is not completely correct.
The length of the sample was hard to determine. While measuring the fibrin sample it was
hard to say whether it might be compressed, extended or in its original length, especially with
the fibrin gels of low concentration fibrinogen.
Once sectioned, the samples were kept in medium till they were used for the measurement.
Two samples were once placed in medium for an hour and weighed before and afterwards to
determine any swelling or shrinking. The two samples had shrunk for about 2 % after an hour.
Therefore it was decided to regard this negligible. This was only done for a sample of 30
mg/ml, the influence on fibrin gels with other concentrations was assumed to be the same but
has not been determined.
The initial Youngs modulus, which has been calculated, is in fact not the real initial modulus
because a pre-load was applied. For the samples with a fibrinogen concentration of 14 mg/ml
and higher a strain of about 3 % was apparent at the pre-load which is quite low. For the
lower concentrations this strain was higher and it is thus less correct to call the calculated
Youngs modulus initial. The applied pre-load was the same for all samples. Better would
have been to apply the same stress for all samples thus to immediately adapt the force to the
area. But it would have been best to apply the same strain for all samples thus adapting the
force to the area and the Youngs modulus for all samples. At first, a few experiments should
be performed to estimate the Youngs modulus and then this estimate could be used in the
setting of the pre-load.
There was no difference in structure seen between the three concentrations. This may be
caused by the freeze-drying, which caused artefacts. The abnormalities seen on the surface of
some samples may have been caused by pollution.
Both samples used for the rheometer were in moderate condition. The first one had a varying
thickness. A constant thickness is a requirement for a rheometer to measure a correct dynamic
modulus. The second one had a tear, which probably influenced the measurement. Varying
the input for the area largely caused the great differences in the found values for the dynamic
modulus. This was done, because the effective area was hard to determine. A possible
explanation for the differences between the found values for the Youngs modulus with the
tensile testing machine and the rheometer is the fact that the way of loading between the two
methods is different. Fibrin gels align when stretched; when compressed there is no alignment
and if shear is then applied, the loading is different than with tensile testing.
It can be said that the protocol that has been set up is very suitable for the testing of fibrin gels
with a high fibrinogen concentration. It would be recommended to use moulds of 5 mm in
width to avoid the influence of sectioning. When testing fibrin gels with a fibrinogen
concentration of 30 mg/ml the thrombin concentration used was 5 IU/ml. It would be
interesting to see what the influence on the Youngs modulus would be when using a higher
concentration of thrombin. Also, the influence of the formation of cross-links by factor XIIIa
on the Youngs modulus would be interesting to investigate.
Acknowledgements
I would like to thank Drs. M. van Lieshout for her very pleasant coaching. She was always
willing to help me with any kind of problem. I also would like to thank Dr. Ir. G. Peters for
his very helpful insights.
References:
1. Julie R. Fuchs MD. et al, Tissue engineering, a 21st century solution to surgical
reconstruction, The annals of thoracic surgery, 72 (2), 577-591, 2001
2. Stefan Jockenhoevel. et al, Fibrin gel advantages of a new scaffold in
cardiovascular tissue engineering, Eur. j. of Cardio-Thoracic surgery, 19 (4), 424430, 2001
3. M. Benkherourou. et al, Quantification and Macroscopic Modeling of the Nonlinear
Viscoelastic Behaviour of Strained gels with Varying Fibrin concentrations, IEEE
Transactions on Biomedical Engineering, 47 (11), 2000
4. Esther A. Ryan. et al, Structural origins of fibrin clot rheology, Biophysical Journal,
77, 2813-2826, 1999
5. D. Sierra. et al, Failure characteristics of multiple-component fibrin-based adhesives,
Department of Biomedical Engineering, University of Alabama at Brimingham, 2001.
6. S. Marshall, Commercial fibrinogen, Autogenous plasma, whole blood and
cyopecipitate for coagulum pyelolithotomy: a comparative study, J. of Urology, 119 ,
1978.
7. J. Velada. et al, Reproducibility of the mechanical properties of Vivostat system
patient-derived fibrin-sealant, Biomaterials, 25 , 2002.
8. Wan. et al, Is the amount of fibrinogen in cryoprecipitate adequate for fibrin glue?
Introducing an improved recycled cryoprecipitate method, Transfusion, 29 , 1989.
9. John B. Walker. et al, The molecular weights, mass distribution, chain composition,
and structure of soluble fibrin degradation products released from a fibrin clot
perfused with plasmin, J. of biological Chemistry, 274 (8), 5201-5212, 1999
10. G. Walker, Teaching Notes, University of Teesside, UK, 2000.
11. C. Macosko, Rheology, principles, measurements and applications, VCH Pubishers,
1994.
Appendix A
deformation of fibrin till break
0.35
0.3
0.25
force (N)
0.2
0.15
0.1
0.05
0
-0.05
-0.5
0.5
1.5
strain
Figure A.1 A sample of fibrin with fibrinogen concentration of 10 mg/ml and an area of 7 *10^-6 m2.
cyclic loading
0.04
0.035
0.03
force (N)
0.025
0.02
0.015
0.01
0.005
0
-0.005
100
200
300
400
500
600
700
800
900
timestep
Figure A.2 Cyclic loading to 20 % strain of a sample of fibrin with fibrinogen concentration of 10
mg/ml and an area of 50 *10^-6 m2
Appendix B
%name: 'force'
%This m-file is to display the four curves of force against
%the strain of one sample
clear all
close all
load A.TRA;
length1=A(:,1);
strain11=(length1-length1(5,1)) /length1(5,1);
%length1 (5,1) contains the length of the sample at the pre-load
%this loop is made to keep out the irregularities seen in the very
%beginning of the testing
r=0;
l1=length(A);
for i=1:l1
strain11(i);
if strain11(i)>0.001
r=r+1;
strain1(r)=strain11(i);
force1(r)=A(i,2);
end
end
%all forces and strain data of test A are being averaged
for i=10:length(strain1)-9
strainaver1(i)=(strain1(i-9)+strain1(i-8)+strain1(i-7)
+strain1(i-6)+strain1(i-5)+strain1(i-4)+strain1(i-3)+strain1(i-2)
+strain1(i-1)+strain1(i)+strain1(i+1)+strain1(i+2)+strain1(i+3)
+strain1(i+4)+strain1(i+5)+strain1(i+6)+strain1(i+7)+strain1(i+8)
+strain1(i+9))/19;
forceaver1(i)=(force1(i-9)+force1(i-8)+force1(i-7)+force1(i-6)
+force1(i-5)+force1(i-4)+force1(i-3)+force1(i-2)+force1(i-1)
+force1(i)+force1(i+1)+force1(i+2)+force1(i+3)+force1(i+4)
+force1(i+5)+force1(i+6)+force1(i+7)+force1(i+8)+force1(i+9))/19;
end
%the same procedure is now done for the three other curves
load B.TRA;
length2=B(:,1);
r=0;
l2=length(B);
for i=1:l2
strain22(i);
r=r+1;
force2(r)=B(i,2);
end
end
%all forces and strain data of test B are being averaged
+strain2(i+9))/19;
end
load C.TRA;
length3=C(:,1);
r=0;
l3=length(C);
for i=1:l3
strain33(i);
r=r+1;
force3(r)=C(i,2);
end
end
%all forces and strain data of test C are being averaged
+strain3(i+9))/19;
end
load D.TRA;
length4=D(:,1);
r=0;
l4=length(D);
for i=1:l4
strain44(i);
r=r+1;
force4(r)=D(i,2);
end
end
%all forces and strain data of test D are being averaged
+strain4(i+9))/19;
end
plot(strainaver1,forceaver1,'y',strainaver2,forceaver2,'m'
,strainaver3,forceaver3,'b',strainaver4,forceaver4,'r')
Appendix C
%name: 'E-modulus'
%This m-file calculates the stress and E-moduli for uniaxial
%extension using the columns generated in the m-file 'force'.
%if planar extension is applied all 'xast'-columns have to be
%changed in 'xasv'-columns. The constant a has to be changed in the value 4
%extension
alfa1(r)=1+strain1(r);
%For uniaxial extension alfa^2 - 1/alfa ('xast') has to be determined and
%for planar extension alfa^2 - 1/alfa^2 ('xasv') has to be determined
for r=1:length(strainaver1)
xast1(r)=(alfa1(r))^2-(1/alfa1(r));
xasv1(r)=(alfa1(r))^2-(1/((alfa1(r))^2));
end
end
end
end
%the constants for the area of the two samples in m^2
areaA=1.897*10^-5;
areaB=2.97*10^-5;
a=3; % constant that calculates E by multiplying G with a
%determining the stress and E

clear t
l1=length(strainaver1);
for t=1:l1
stress1(t)=forceaver1(t)*((1+strainaver1(t)))/areaA;
end
clear t
for t=1:(l1-1)
E1(t)=a*(stress1(t+1)-stress1(t))/(xast1(t+1)-xast1(t));
end
clear t
for t=1:l1
end
clear t
for t=1:(l1-1)
end
clear t
for t=1:l2
end
clear t
for t=1:(l2-1)
end
clear t
for t=1:l3
stress3(t)=forceaver3(t)*((1+strainaver3(t)))/areaB;
end
clear t
for t=1:(l3-1)
end
clear t
for t=1:l4
stress4(t)=forceaver4(t)*((1+strainaver4(t)))/areaB;
end
clear t
for t=1:(l4-1)
end
%plotting of stress
plot(xast1,stress1,'y',xast2,stress2,'m',xast3,stress3
,'b',xast4,stress4,'r')
xlabel('alfa^2 - 1/alfa')
title('stress (N/m^2)')
pause
%plotting of Young's-moduli
plot(xast1,E1(1:l1-1),'y',xast2,E2(1:l2-1),'m',xast3,E3(1:l3-1)
,'b',xast4,E4(1:l4),'r')
ylabel('Youngs-modulus')
pause
%taking all data together
clear r
clear t
b=25; %constants are less when less data are present in file than 2500
c=25;
d=25;
e=25;
f=0;
for i= b:l1
if xast1(i)<0.25 %when determining the strain at alfa^2 - 1/alfa = 0.6
%then 0.25 turns into 0.7
f=f+1;
end
end
g=0;
for i= c:l3
if xast2(i)<0.25
g=g+1;
end
end
h=0;
for i= d:l3
if xast3(i)<0.25
h=h+1;
end
end
j=0;
for i= b:l4
if xast4(i)<0.25
j=j+1;
end
end
Ex=[xast1(b:f) xast2(c:g) xast3(d:h) xast4(e:j)];

Ey=[E1(b:f) E2(c:g) E3(d:h) E4(e:j)];
[x i]=sort(Exx);
y=Eyy(i);
plot(x,y)
ylabel('Youngs-modulus fitted')

Influence of Fibrinogen Concentration On Young's Modulus in Fibrin Gels

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Influence of fibrinogen

concentration on the Youngs

Supervisors: Drs. M. van Lieshout, Dr. Ir. G. Peters

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

Department of Biomedical Engineering 2

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

Department of Biomedical Engineering 3

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

Department of Biomedical Engineering 4

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

Department of Biomedical Engineering 5

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

1.1.2 Formation of fibrin network

Figure 1.2 The formation of fibrin network [9].

Fig 1.3 Spring and Dashpot models for visco-elasticity [10].

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

If a strain is applied to a visco-elastic material there is an instantaneous response (elastic) and

G = G '+iG ' '

G = (G ' 2 + G ' ' 2 )

where F is the deformation gradient tensor.

where is the ratio of deformed to undeformed length.

Department of Biomedical Engineering 7

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

For an incompressible material

volume = a1x1 = a1* x1*

In the limit of small strain

which is in the linear region of figure 1.4.

The Youngs modulus can be defined as

Department of Biomedical Engineering 8

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

1.2.1.2 Planar extension

Department of Biomedical Engineering 9

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

2. Materials and Methods

Figure 2.1 The two-component fibrin sealant.

Department of Biomedical Engineering 10

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

2.1 Fibrin gel preparation

2.2 Tensile testing

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

2.3 Data processing

2.4 SEM preparation

2.5 Rheology testing

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

Department of Biomedical Engineering 13

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

Department of Biomedical Engineering 14

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

Youngs' modulus (Pa)

Figure 3.4 Linear fit through all data up to 2-1/ =0.25.

The initial Youngs modulus here is 9.574 kPa.

Department of Biomedical Engineering 15

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

Young's modulus (Pa)

Department of Biomedical Engineering 16

Influence of fibrinogen concentration in fibrin gels on Youngs modulus

Initial Young's modulus

Young's modulus (Pa)

Young's modulus and standard deviation

concentration fibrinogen (mg/ml)

Figure 3.6 Initial averaged Youngs modulus and standard deviation

Young's modulus at alfa^2 - 1/alfa =0.6

Young's modulus (Pa)