Mechanical Properties of Collagen Fibrils

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    EDST - UL Ecole Doctorale des Sciences

    et de Technologie Universit Libanaise

    Nanotechnologie et Nano-Biomcanique des

    Systmes 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

    2 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

    3 Sarah Hussein Master TIS

  • O U T L I N E S

    INTRODUCTION METHODOLOGIES RESULTS CONCLUSIONS

    1 2 3 4

    4 Sarah Hussein Master TIS

  • 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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    Sarah Hussein Master TIS

  • 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

    7 Sarah Hussein Master TIS

  • 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 Youngs 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 4C

    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

    10 Sarah Hussein Master TIS

  • 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

    Sarah Hussein Master TIS

  • Nano Indentation By AFM

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    M A T E R I A L S & M E T H O D S

    Sarah Hussein Master TIS

  • 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

    Sarah Hussein Master TIS

  • RESULTS

    Youngs Modulus Anisotropy of Collagen Fibrils Fibrils Dehydration & Tip Shape Accuracy of Reduced modulus

    3

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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 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

    19 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

    20 Sarah Hussein Master TIS

  • O U T L I N E S

    INTRODUCTION METHODOLOGIES RESULTS CONCLUSIONS

    1 2 3 4

    21 Sarah Hussein Master TIS

  • 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 ~1520% 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

    26 Sarah Hussein Mas