mechanical properties of collagen fibrils
TRANSCRIPT
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EDST - UL Ecole Doctorale des Sciences
et de Technologie Université Libanaise
Nanotechnologie et Nano-Biomécanique des
Systèmes Biologiques Complexes
Submitted to Dr. Karim EL KIRAT February 19th, 2016
Sarah Hussein Master TIS
A G E N D A
Article 1 § Authors: Marco P. E. Wenger Laurent Bozec Michael A. Horton Patrick Mesquidaz § Journal: Biophysical Journal Volume 93 August 2007
Article 2 § Authors: Colin A. Grant David J. Brockwell Sheena E. Radford Neil H. Thomson § Journal: Biophysical Journal Volume 97 December 2009
“Mechanical Properties of Collagen Fibrils”
“Tuning the Elastic Modulus of Hydrated Collagen Fibrils”
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A G E N D A
Article 1 § Authors: Marco P. E. Wenger Laurent Bozec Michael A. Horton Patrick Mesquidaz § Journal: Biophysical Journal Volume 93 August 2007
Article 2 § Authors: Colin A. Grant David J. Brockwell Sheena E. Radford Neil H. Thomson § Journal: Biophysical Journal Volume 97 December 2009
“Mechanical Properties of Collagen Fibrils”
“Tuning the Elastic Modulus of Hydrated Collagen Fibrils”
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O U T L I N E S
INTRODUCTION METHODOLOGIES RESULTS CONCLUSIONS
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INTRODUCTION
¨ Context ¨ Purpose 1�
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I N T R O D U C T I O NContext
q Collagen is a very abundant structural protein in mammals
q It is a major component of connecting tissue, skin, bone, cartilage and tendons
q The development of collagen model is important
q There is a lack of convincing and comprehensive structural model for fibrils
q The biological function of collagen lies in its mechanical properties
q There is a necessity to determine these mechanical properties at different scales
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I N T R O D U C T I O NContext (Cont.)
q These information are important to explain the macroscopic biophysics of
different tissues
q In addition, it contribute to the understanding of the microscopic structure of
collagen fibrils
q Conventional macroscopic technical tools are not applicable to Nano fibrils
q Other techniques are used, the Nano scale indentation is one such a technique
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I N T R O D U C T I O NPurpose
“To determine the mechanical properties of individual type I collagen fibrils of rat tail tendon in
air & at room temperature”
“The Young’s modulus is determined quantitatively using sharp AFM tip in combination
with the Olivier and Pharr indentation model”
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MATERIALS & METHODS
¨ Sample preparation ¨ AFM Imaging ¨ Nano-Indentation by AFM
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M A T E R I A L S & M E T H O D SSample Preparation
q Type I collagen fibrils extracted from rat tail tendons and stored at 4°C
q The sample is sectioned with a scalpel and washed in deionized water
q A microscope glass slide was used as sample substrate
q It was cleaned with DI water in an ultrasonic bath and rinsed with ethanol and
dried in a stream of nitrogen
q A few samples deposited on the glass slide and smeared out using tweezers
q The sample was then dried in a gentle stream of nitrogen
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AFM Imaging
q AFM imaging and indentation of
collagen fibrils using a Nanowizard AFM
q All measurements were taken in air and
at room temperature
q Aluminum-coated, silicon AFM tips of
150 kHz resonance frequency and 4.5
N/m nominal spring constant is used
q Cantilever chosen to match stiffness of
collagen for optimizing sensitivity & SNR
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M A T E R I A L S & M E T H O D S
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Nano Indentation By AFM
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M A T E R I A L S & M E T H O D S
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Nano Indentation By AFM (Cont.)
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M A T E R I A L S & M E T H O D S
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RESULTS
¨ Young’s Modulus ¨ Anisotropy of Collagen Fibrils ¨ Fibrils Dehydration & Tip Shape ¨ Accuracy of Reduced modulus
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R E S U L T SAnisotropy of Collagen Fibrils
q Two fibrils used from the same sample,
similar in diameter, and aligned
perpendicular to each other
q The same AFM tip used for experiments
and its orientation was not changed
q Reduced modulus was found to be in
the range from 5 GPa to 11.5 Gpa
q Non-uniform shape of the imprints
indicates different material properties in
longitudinal and transversal directions
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R E S U L T SFibrils Dehydration & Tip Shape
q Tendency of the Er to increase slightly
from 6 GPa to 7.5 GPa over three days
q The indentation depth decreases from
3.5 nm to 2.5 nm
q Dehydrat ion of co l lagen f ibr i l s
contributes with 30% to the broad
distribution of fibril moduli
q Tip shape by AFM imaging revealed a
spherical tip apex
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R E S U L T SAccuracy of The Reduced Modulus Determination
q The accuracy of reduced modulus depends on the experimental accuracy of the
determination of the contact area (A) and of the fibril stiffness (SF)
q Stiffness is directly proportional to the cantilever spring constant: 5% error
q For contact area, AFM tip radius and indentation depth is considered : 20% for
the radius determination and 10% for the indentation depth à 30%
q Another possible source of error of contact area are attractive interfacial forces
between AFM tip and sample surface
q The effects discussed above are assumed to be less relevant in the present work
and were not taken into account
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CONCLUSIONS
¨ Brief Summary 4�
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C O N C L U S I O N SBrief Summary
q The great advantage of Nano-Indentation is the small size of the indenter
q Using an indenter with tip apex smaller than the collagen fibril diameter,
indentation caused small imprints
q The non-uniform shape of these imprints indicates an anisotropic material
structure of collagen fibrils
q This anisotropy can be neglected as its contribution to the overall experimental
error is smaller than the variation of the stiffness upon fibril dehydration
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A G E N D A
Article 1 § Authors: Marco P. E. Wenger Laurent Bozec Michael A. Horton Patrick Mesquidaz § Journal: Biophysical Journal Volume 93 August 2007
Article 2 § Authors: Colin A. Grant David J. Brockwell Sheena E. Radford Neil H. Thomson § Journal: Biophysical Journal Volume 97 December 2009
“Mechanical Properties of Collagen Fibrils”
“Tuning the Elastic Modulus of Hydrated Collagen Fibrils”
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O U T L I N E S
INTRODUCTION METHODOLOGIES RESULTS CONCLUSIONS
1� 2� 3� 4�
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INTRODUCTION
¨ Context ¨ Purpose 1�
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I N T R O D U C T I O NContext
q Huge importance of collagen fibrils
q Collagen has a structure of triple helix of left-handed polypeptide chains coming
together to form right-handed twisted molecule (tropocollagen)
q The generalized well-known formula for collagen amino acid sequence is Gly-X-
Y, where X is proline (Pro) and Y a hydroxyproline (Hyp) residue
q Glycine, which occurs at every third residue, is found at the center of the coiled
peptide chain to allow a close packing of the triple helix
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I N T R O D U C T I O NContext (Cont.)
q Charged residues make up ~15–20% of residues in tropocollagen, and ~40% of
Gly-X-Y sequences contain at least one charged residue
q Electrostatic interactions are of great importance for the stability of the triple-
helical conformation with tropocollagen q Studies have shown that hydrated collagen fibrils under bulk aqueous solution
conditions have a reduction in modulus compared to the anhydrates
q Measured hydrated shear modulus changes very little when a cross-linker is
applied to collagen fibrils
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I N T R O D U C T I O NPurpose
“To highlights the range of elastic response of collagen type I fibrils that can be achieved in liquid by
altering the environment” &
“To discuss the finding that the mechanical properties of hydrated collagenous fibrils can be tuned by adding salts, by changing the solution pH, or by
changing the solvent”
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MATERIALS & METHODS
¨ AFM & Nano-Indentation 2�
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M A T E R I A L S & M E T H O D SAFM & Nano-Indentation
q AFM imaging and force measurements were made using silicon nitride
cantilevers with integral tips with spring constants of the order k ~ 0.3 N/m
q Force volume (FV) imaging was carried out on isolated fibrils using arrays of 50
� 50 pixels, with each pixel representing a single force-distance measurement
q The built-in software is used to calculate both the cantilever spring constant and
the reduced elastic modulus (Er), which uses a Hertzian contact theory
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RESULTS
¨ Effect of Fibril Swelling in Aqueous buffer
¨ Effect of Salt Concentration
¨ Effect of Altering The Cation Species
¨ Effect of Lowering pH ¨ Effect of Ethanol
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R E S U L T SEffect of Fibril Swelling in Aqueous buffer
q Measurements of the same collagen fibrils
demonstrate that stable AFM imaging can be
achieved in liquids
q Swelling occurs in the hydrated form but does
not change significantly among different
conditions of salt and pH
q Collagen fibril mechanics are influenced by the
liquid phase of this biocomposite material
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R E S U L T SEffect of Salt Concentration
q NaCl was added to the 100 mM sodium
phosphate buffer to concentrations up to 1 M
q Modulus value showed no appreciable
change up to 500 mM NaCl, but 2.3 times
increase in modulus detected at 1 M NaCl
q Imaging of the fibril in 1 M NaCl showed no
noticeable change in the morphology
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R E S U L T SEffect of Altering The Cation Species
q Various monovalent chloride salt solutions
were used at concentrations up to 1 M
q Cation species does not influence final value
of elastic modulus at 1 M monovalent
chloride salt
q Increase in modulus at higher salt
concentration appears to be related to ionic
strength of solution surrounding collagen
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R E S U L T SEffect of Lowering pH
q Effect on modulus of adding salt (1 M KCl)
greatly enhanced when the pH was lowered
q The combined effects of lowering pH to 5
and increasing salt concentration to 1 M
raises the modulus by 10-fold compared
with its value at pH 7 with no salt
q Increasing the ionic strength gradually
through the use of two different potassium
acetate buffers and and increasing amounts
of KCl, gave a gradual increase in the
measured modulus
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R E S U L T SEffect of Ethanol
q Freshly prepared collagen fibril were placed
under an increasing concentration of ethanol
in 100 mM sodium phosphate
q Modulus steadily increases with increasing
ethanol concentration up to 50%
q Large increase in modulus was found when
scanning medium increased to 100% ethanol
q Indentation depths measured from the FV
analysis in 100% ethanol are lower than
those measured in buffer
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CONCLUSIONS
¨ Brief Summary 4�
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C O N C L U S I O N SBrief Summary
q When salt concentration or ionic strength is increased, hydration or solvation forces dominate the response
q When the pH is lowered, ion pair interactions would seem the most likely to dominate
q It is also conceivable that hydrophobic effects play a role in all these scenarios but likely at a lower magnitude than the hydrophilic forces described in each case
q A better understanding of which forces modulate the elastic response when one parameter is changed should aid rational design of new materials based on collagen and analogous synthetic peptides
q All these effects appear to be fully reversible, which may indicate that it is possible to modulate tissue elasticity in vivo by directed therapeutics, whether the tissue in question is natural or bioengineered implants
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THANK YOU
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