mechanical properties of collagen fibrils

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1 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 19 th , 2016 Sarah Hussein Master TIS

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Page 1: Mechanical Properties of Collagen Fibrils

1

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

Page 2: Mechanical Properties of Collagen Fibrils

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”

2 Sarah Hussein Master TIS

Page 3: Mechanical Properties of Collagen Fibrils

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”

3 Sarah Hussein Master TIS

Page 4: Mechanical Properties of Collagen Fibrils

O U T L I N E S

INTRODUCTION METHODOLOGIES RESULTS CONCLUSIONS

1� 2� 3� 4�

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Page 5: Mechanical Properties of Collagen Fibrils

INTRODUCTION

¨  Context ¨  Purpose 1�

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Page 6: Mechanical Properties of Collagen Fibrils

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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Sarah Hussein Master TIS

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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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Page 8: Mechanical Properties of Collagen Fibrils

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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Page 9: Mechanical Properties of Collagen Fibrils

MATERIALS & METHODS

¨  Sample preparation ¨  AFM Imaging ¨  Nano-Indentation by AFM

2�

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Page 10: Mechanical Properties of Collagen Fibrils

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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Page 11: Mechanical Properties of Collagen Fibrils

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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Page 12: Mechanical Properties of Collagen Fibrils

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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Page 13: Mechanical Properties of Collagen Fibrils

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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Page 14: Mechanical Properties of Collagen Fibrils

RESULTS

¨  Young’s Modulus ¨  Anisotropy of Collagen Fibrils ¨  Fibrils Dehydration & Tip Shape ¨  Accuracy of Reduced modulus

3�

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Page 15: Mechanical Properties of Collagen Fibrils

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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Page 16: Mechanical Properties of Collagen Fibrils

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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Page 17: Mechanical Properties of Collagen Fibrils

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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Page 18: Mechanical Properties of Collagen Fibrils

CONCLUSIONS

¨  Brief Summary 4�

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Page 19: Mechanical Properties of Collagen Fibrils

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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Page 20: Mechanical Properties of Collagen Fibrils

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”

20 Sarah Hussein Master TIS

Page 21: Mechanical Properties of Collagen Fibrils

O U T L I N E S

INTRODUCTION METHODOLOGIES RESULTS CONCLUSIONS

1� 2� 3� 4�

21 Sarah Hussein Master TIS

Page 22: Mechanical Properties of Collagen Fibrils

INTRODUCTION

¨  Context ¨  Purpose 1�

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Page 23: Mechanical Properties of Collagen Fibrils

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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Page 24: Mechanical Properties of Collagen Fibrils

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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Page 25: Mechanical Properties of Collagen Fibrils

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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Page 26: Mechanical Properties of Collagen Fibrils

MATERIALS & METHODS

¨  AFM & Nano-Indentation 2�

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Page 27: Mechanical Properties of Collagen Fibrils

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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Page 28: Mechanical Properties of Collagen Fibrils

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

3�

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Page 29: Mechanical Properties of Collagen Fibrils

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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Page 30: Mechanical Properties of Collagen Fibrils

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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Page 31: Mechanical Properties of Collagen Fibrils

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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Page 32: Mechanical Properties of Collagen Fibrils

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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Page 33: Mechanical Properties of Collagen Fibrils

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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Page 34: Mechanical Properties of Collagen Fibrils

CONCLUSIONS

¨  Brief Summary 4�

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Page 35: Mechanical Properties of Collagen Fibrils

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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Page 36: Mechanical Properties of Collagen Fibrils

THANK YOU

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