1. Centre for Doctoral
Training in Medical and
Biological Engineering
Andres Barco1, J Fisher1, Hazel Fermor1, Robert P. W. Davies2, E Ingham1
1 Institute of Medical and Biological Engineering, University of Leeds, United Kingdom. 2 Department of Oral Biology, School of Dentistry, University of Leeds, United Kingdom.
mn09ab@leeds.ac.uk
Rheological properties of peptide
based hydrogel-
glycosaminoglycan mixtures
• In previous studies, Fourier transform infrared
spectroscopy (FTIR) revealed the effect that
increasing the molar ratio of GAG and salt
concentration had on the percentage of β-sheet
formation. Peptide-GAG mixtures, P11-4 and P11-8
showed the highest β-sheet formation in both Na+
salt solutions and molar ratios of GAG (below).
• This was complemented by images from transition
electron microscopy (TEM) which revealed bundles
of aggregates with a high percentage of junction
points (right).
• All samples were investigated in two
different salt solutions at a temperature
of 37oC
• Peptide alone, peptide in combination
with chondroitin sulphate at two different
molar ratios and chondroitin sulphate
alone were investigated using rheology.
• An amplitude sweep at 1Hz and 20Hz
was run to determine a suitable strain
value within the linear viscoelastic region
(LVER).
• This strain value was used to run a
frequency sweep across a range of
frequencies (1-20Hz) to determine the
elastic and viscous modulus of each
material.
The aims of this study were to investigate
the mechanical properties of self-assembling
peptide hydrogels based on three peptides
of the P11 series in combination with varying
molar ratios of chondroitin sulphate using
rheology.
2. Progress
1. Introduction 3. Aim
4. Methods
Summary & Future work
• Healthy articular cartilage has a complex network of
glycosaminoglycan (GAG) aggregates which is essential for the
correct tissue behaviour [1].
• A net depletion of glycosaminoglycan’s (GAGs) in osteoarthritic
cartilage results in the loss of mechanical properties and function in
vitro [2,3,4].
• Studies to restore GAGs to GAG depleted articular cartilage with
self-assembling peptides (SAPs) and chondroitin sulphate, have
shown promising results in the restoration biomechanical properties
[4].
• A common experimental method used to investigate the mechanical
properties of these hydrogel mixtures is rheology.
• An essential understanding of the mechanical properties of the
peptide hydrogels with and without GAGs and the underlying
molecular mechanisms, is fundamental in determining whether these
biomaterials are potentially suitable for regenerative medicine
applications.
Degeneration process InterventionNatural Cartilage
Cartilage
degeneration
Regeneration
Peptide
Monomer
Anionic contrast
agent
Healthy Cartilage
(more GAG)
Early Stage OA
(less GAG)
Late Stage OA
(very few GAG)
GAG molecule
GAG
solution
1720 1700 1680 1660 1640 1620 1600 1580
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6 (A)
1690 - 1682 cm-1
1630 - 1613 cm-1
Absorbance
Wavelength (cm-1
)
P11-4 alone
P11-4 at 1:64
P11-4 at 1:16
P11-8 alone
P11-8 at 1:64
P11-8 at 1:16
P11-12 alone
P11-12 at 1:64
P11-12 at 1:16
-sheet %
- 93.8%
- 51%
- 70%
- 42.9%
- 60%
- 54%
- 21.5%
- 29%
- 0%
1720 1700 1680 1660 1640 1620 1600 1580
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1690 - 1682 cm-1
Absorbance
Wavelength (cm-1
)
P11-4 alone
P11-4 at 1:64
P11-4 at 1:16
P11-8 alone
P11-8 at 1:64
P11-8 at 1:16
P11-12 alone
P11-12 at 1:64
P11-12 at 1:16
1630 - 1613 cm-1
(B)
-sheet %
- 79.3%
- 69%
- 91%
- 55.1%
- 40%
- 27%
- 51.6%
- 0%
- 0%
10 µm 10 µm 10 µm
10 µm 10 µm 10 µm
230 mM
130 mM
P11-4 @ 230 mM // 1: 64 P11-4 @ 230 mM // 1: 16
P11-4 @ 130 mM // 1: 64 P11-4 @ 130 mM // 1: 16
P11-4 @ 230 mM
P11-4 @ 130 mM
A B C
D FE
10 µm 10 µm 10 µm
10 µm 10 µm 10 µm
230 mM
130 mM
P11-8 @ 230 mM // 1: 64 P11-8 @ 230 mM // 1: 16
P11-8 @ 130 mM // 1: 64 P11-8 @ 130 mM // 1: 16
P11-8 @ 230 mM
P11-8 @ 130 mM
A B C
D FE
10 µm 10 µm 10 µm
10 µm 10 µm 10 µm
230 mM
130 mM
P11-12 @ 230 mM // 1: 64 P11-12 @ 230 mM // 1: 16
P11-12 @ 130 mM // 1: 64 P11-12 @ 130 mM // 1: 16
P11-12 @ 230 mM
P11-12 @ 130 mM
A B C
D FE
Figure 1: Fitted IR amide I’ band of P11-4, P11-8 & P11-12 at 10
mg.ml-1 in presence of 130 mM (A) and 230 mM (B) Na+ salt
solution at varying GAG molar ratios. The β-sheet percentage
was calculated by adding the total area of the peaks showing β-
sheet and then dividing them by the areas of all the individual peaks
combined for each graph and multiplying by 100. The β-sheet
regions are defined by the peaks in the wavelength region of 1630-
1613 cm-1 and 1690-1682 cm-1.
Figure 2: Morphology of the P11-4, P11-8 and P11-12
peptides in the presence of two Na+ salt solutions at
varying GAG molar ratios (1:16 and 1:64) by TEM.
Magnification of 500.Individual scale bars are shown for
each image.
• All variables (peptide, salt concentration,
and chondroitin sulphate molar ratio)
had a significant effect on the
mechanical properties of the hydrogels
at 1 Hz.
• In all samples elastic modulus was
higher than viscous modulus,
suggesting solid-like behaviour.
• Two of the peptide-hydrogel mixtures,
P11-4 & P11-8, showed greater
mechanical strength in both salt
solutions and molar ratios when
compared P11-12.
1
10
100
1000
10000
100000
P11- 4 // 1:16P11- 4 // 1:64
ShearModulus,G',G''(Pa)
Shear Mod (Elas) Shear Mod (Elas)
Shear Mod (Vis) Shear Mod (Vis)
P11- 4 alone
130mM Na+
Salt Solution
P11- 4 // 1:16P11- 4 // 1:64P11- 4 alone
230mM Na+
Salt Solution
1
10
100
1000
10000
100000
ShearModulus,G',G''(Pa)
Shear Mod (Elas) Shear Mod (Elas)
Shear Mod (Vis) Shear Mod (Vis)
P11- 8 // 1:16P11- 8 // 1:64P11- 8 alone
130mM Na+
Salt Solution
P11- 8 // 1:16P11- 8 // 1:64P11- 8 alone
230mM Na+
Salt Solution
1
10
100
1000
10000
100000
ShearModulus,G',G''(Pa)
Shear Mod (Elas) Shear Mod (Elas)
Shear Mod (Vis) Shear Mod (Vis)
P11- 12 // 1:16P11- 12 // 1:64P11- 12 alone
130mM Na+
Salt Solution
P11- 12 // 1:16P11- 12 // 1:64P11- 12 alone
230mM Na+
Salt Solution
5. Results
Figure 3: The effect of varying the Na+ ion concentration (130 mM
or 230 mM) and GAG molar ratio (1:64 or 1:16) on the mechanical
properties of the P11-4, P11-8 and P11-12 gels.
The shear moduli were all taken from the frequency sweep test at 1Hz.
Data is presented as the mean (n=3) ± 95% confidence intervals. Data
was analysed using a three-way analysis of variants and statistical
significance was determined at p<0.05. This showed that all the
independent variables alone (peptide choice, GAG molar ratio and salt
solution) had a significant effect on the mechanical properties across all
three peptides (p=0). Their combined effects showed that peptide choice
in combination with the salt solution had no significant effect on the
overall mechanical properties of all three peptides (p=0.065). For the
combined effects of peptide choice, GAG molar ratio and salt solution,
there was a significant effect observed in the mechanical properties of
all three peptide gels (p=0).
• P11-12 peptide-hydrogel mixtures
formed weak gels (elastic modulus
below ~2000 Pa).
• P11-4 peptide-hydrogel mixtures formed
stronger gels (elastic modulus between
~5000-10,000 Pa).
• P11-8 peptide-hydrogel mixtures formed
the strongest gels (elastic modulus
between ~8000-20,000 Pa), which is
relatively high for a gel made of soft
matter.
• Therefore, P11-4 & P11-8 peptide-
hydrogel mixtures have been chosen
for further investigation in
glycosaminoglycan depleted model
tissues to determine whether they can
restore their mechanical properties, via
a minimally invasive injection
procedure.
[1] Dudhia J, Cell Mol Life Sci 2005;62(19-20):2241-2256 [2] Otsuki S, Nakajima M, Lotz M, Kinoshita M, J Orthop Res 2008;26(9):1194-1198. [3] Mankin HJ, Lippiell.L, Journal of Clinical Investigation 1971;50(8):1712.
[4] Katta J, Stapleton T, Ingham E, Jin ZM, Fisher J, Proc. Inst. Mech Eng Part H 2008;222(H1):1-11. [5] http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-17/CB17.html [6] http://ars.els-cdn.com/content/image/1-
s2.0-S1063458409002441-gr1.jpg
References:
[5]