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TRANSCRIPT
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 [email protected]
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
Abs
orba
nce
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
Abs
orba
nce
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 // 1:16 P11- 4 // 1:64
She
ar M
odul
us, G
', G
'' (P
a)
Shear Mod (Elas) Shear Mod (Elas) Shear Mod (Vis) Shear Mod (Vis)
P11- 4 alone
130mM Na+ Salt SolutionP11- 4 // 1:16 P11- 4 // 1:64 P11- 4 alone
230mM Na+ Salt Solution
1
10
100
1000
10000
100000
She
ar M
odul
us, G
', G
'' (P
a)
Shear Mod (Elas) Shear Mod (Elas) Shear Mod (Vis) Shear Mod (Vis)
P11- 8 // 1:16 P11- 8 // 1:64 P11- 8 alone
130mM Na+ Salt SolutionP11- 8 // 1:16 P11- 8 // 1:64 P11- 8 alone
230mM Na+ Salt Solution
1
10
100
1000
10000
100000
She
ar M
odul
us, G
', G
'' (P
a)
Shear Mod (Elas) Shear Mod (Elas) Shear Mod (Vis) Shear Mod (Vis)
P11- 12 // 1:16 P11- 12 // 1:64 P11- 12 alone
130mM Na+ Salt SolutionP11- 12 // 1:16 P11- 12 // 1:64 P11- 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]