Hierarchical Approaches to Investigating Tissue

Micromechanics

Hazel Screen

School of Engineering & Materials Science,Queen Mary, University of London

6th November 2008

Connective Tissue Function & Health

• Connective tissues = structural support

• “cartilage once destroyed, is not repaired” Hunter. W, 1743

• Normal healing mechanisms are unavailable to damaged connective tissues

Investigating Tissue Micromechanics

1. Understanding tissue structure and how to help protect it from damage

2. Understand how to facilitate repair in damaged tissue

How to Facilitate Tissue Repair

Chemical cues:

Growth factorsNutrients

Mechanical cues:

Fluid flowPressureDeformation

• Regulates normal tissue homeostasis• Implicated in pathological processes• Implicated in repair processes• Harness it for tissue engineering??

MechanicalLoading(in vitro)(in vivo)

Altered Cell Response

ProliferationMatrix synthesis

Matrix degradationCell/matrix orientation

Mechanotransduction

The Hierarchy of Mechanobiology

Body mechanics

Joint mechanics

Tissue mechanics

Cell mechanics

Protein mechanics

The Hierarchy of Mechanobiology

Body mechanics

Joint mechanics

Tissue mechanics

Cell mechanics

Protein mechanics

• How does the tissue hierarchy control mechanical properties?

• How does the material deform:

• How are strains transferred to the cells?

Investigate the local mechanical environment as

the mechanotransduction stimulus of interest

Tissue Composition & Mechanics

Tissue Composition & Tissue Mechanics

Articular cartilage Tendon / ligament Skin

Aortic valve

In Situ Analysis Techniques

Screen et al. (2003) Biorheol. 40, 361-8

Stepper Motor

Heater PadsMicroscopeObjective Lens

Grips

Specimen Medium

Coverslip

Screen et al. (2004) J. Eng. Med. 218, 109-19

• Custom designed rig for location on confocal microscope

• Enables tensile / compressive loading of viable tissue

samples

• Use range of matrix & cell stains to visualise matrix

components during loading

Tendon Structure

TendonFascicle

Endotendon

TenocyteFibre

Crimp waveform

Fibril

Crimping

Microfibril

Tropocollagen

1.5 3.5 50-500 10-50 50-400 500-2000nm m

Multi-level fibre composite Considered simple collagen tissue to study

Tendon Extension Mechanisms

uFibre Extension

00.20.40.60.8

11.21.41.61.8

2

0 2 4 6 8Applied Strain (%)

Wit

hin-

grou

p st

rain

(%

)

Fibre Sliding

0

1

2

3

4

5

0 2 4 6 8Applied Strain (%)

Bet

wee

n g

rou

p d

isp

lace

men

t (%

ap

pli

ed d

isp

lace

men

t)

v L

u

Fibre Extension

v L

Fibre Sliding

Screen et al. (2004) J. Strain 40:4, 157-163

Tendon Extension Mechanisms

Collagen molecule

Fibril

Fibre

Fascicle

Tendon Extension Mechanisms

Shearing/

Sliding

Extension

rotation

Collagen molecule

Fibril

Fibre

Fascicle

What controls the fibre composite behaviour?

• Non-Collagenous Matrix

Shape Molecule

Scott (2003) J. Physiol. 553; 335-343

Scott & Thomlinson (1998) J. Anat. 192; 391-405

• Decorin: Binds around collagen fibrils

Screen et al (2005) Ann Biomed Eng 33; 1090-1099

0

0.5

1

1.5

2

2.5

0 100 200 300 400 500Time (secs)

For

ce (

N)

0

0.5

1

1.5

2

2.5

0 100 200 300 400 500

Time (secs)

For

ce (

N)

Understanding Viscoelasticity

• Very rapid relaxation ; Total relaxation < 60 secs

• Highly viscous tissue

Direct tests Incremental tests

8%

2%

4%

6%8%

Gross mechanical properties:

Confocal Images – Stress Relaxation

Confocal Images – Stress Relaxation

Fibre Relaxation

Fibre Siding

collagen fibre

tenocyte nuclei

Applied Extension = L

Confocal Images – Stress Relaxation

y = -0.0002x + 0.0151

R2 = 0.0034

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0 20 40 60

Time (secs)

y = -0.0029x - 0.214

R2 = 0.6649

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0 20 40 60

Time (secs)

Pe

rce

nta

ge

fib

re r

ela

xatio

n (

%)

Pe

rce

nta

ge

be

twe

en

-fib

re r

ela

xatio

n (

%)

Fibre Relaxation Fibre Sliding

TYPICAL DATA:4 % Applied Strain

Confocal Images – Stress Relaxation

Bet

wee

n-fib

re d

ispl

acem

ent (

m)

-2.5

-2

-1.5

-1

-0.5

0

Applied Strain (%)

Fibre Relaxation

1% 2% 4% 6% 8%

Fibre Sliding

Fib

re r

elax

atio

n (

m)

-0.06

-0.05

-0.04

-0.03

-0.02

-0.01

0

0.01

0.02

Applied Strain (%)

1% 2% 4% 6% 8%

Confocal Images – Stress Relaxation

How does this affect the cells?

We now have some understanding of the mechanisms of extension & relaxation:

What does this mean for the local strain environment throughout the sample and

surrounding the cells?

Finite Element Approach

Track coordinates of every cell

Construct a Delaunay mesh of triangle elements

Monitor deformation & strain in each element during relaxation

Important coordinates into Matlab

S Evans - Cardiff University

x

y

Finite Element Approach

X displacement Y displacement

Displacements

Y displacementX displacement

x

y

Shear strain

Relaxation Strains

x

y

X strain Y strain

Huge variability in response Strain seems random

Relaxation Strains

x

y

x strains

0

5

10

15

20

25

-0.4 -0.3 -0.2 -0.2 -0.1 0 0.08 0.16 0.24 0.32 0.4

Strain

0

5

10

15

20

25

-0.4 -0.3 -0.2 -0.2 -0.1 0 0.08 0.16 0.24 0.32 0.4

Strain

y strains

0

5

10

15

20

25

-0.4 -0.3 -0.2 -0.2 -0.1 0 0.08 0.16 0.24 0.32 0.4

Shear strain

shear strains

Predominantly negative= compression

Range positive & negative= Fibre sliding

Wide range of shear strains

Relaxation Behaviour

• Relaxation strains far exceed the initial applied strain

• Values are both positive and negative

• Monitoring deformation of each triangle

• Significant sliding between cells on different fibres

• Sliding creates large shear strain in matrix (on cells)

Loading Direction:

Transverse Direction:

• More uniform response & predominantly negative strains

• Water movement out of inter-fibre spacing

Cell Perspective

Cell processes link adjacent rows of cells:

• Large deflections (y strains)

• Compressive loading of cells

(x strains)

Other Hierarchical ChangesTendonFascicle

Endotendon

TenocyteFibre

Crimp waveform

Fibril

CrimpingMicrofibril

Tropocollagen

1.5 3.5 50-500 10-50 50-400 500-2000nm m

Confocal focus

X-ray synchrotron scattering Himadri Gupta (Max Plank)

Synchrotron X-ray Scattering

ESRF BL ID2Peter Boesecke

(Grenoble)

CC

D X

– r

ay

dete

ctor X - ray

Load cell

Small angle X – ray scattering (SAXS) setup

2/D

Microtensile testerMax load 250 g – 12 kg

Strain measured with video extensometry(NON-contact)

Fibril Strain During Relaxation

Two time constants + , -

021

021

expexp

expexp

στ

tΔσ

τ

tΔσσ(t)

ετ

tε

τ

tε(t)ε FFFF

General Form

Stre

ss (

MPa

)Fi

bril

str

ain

(%)

60

50

40

30

20

10

0

0 100 200 300

0 100 200 300

Time (Seconds)

Time (Seconds)

2.5

2.0

1.5

1.0

Fitting Data:+

σ & +ε ≤ 10 s

-σ & -

ε ≥ 50 s

Fitting ‘ε’ constants to ‘σ’ ?

0 100 200 300

fib

ril

stra

in [

%]

1.42

1.44

1.46

1.48

1.50

1.52

1.54

1.56

1.58

0 100 200 300

stre

ss [

MP

a]

14

16

18

20datafitfit, ef tcs

Fibril relaxation & stress relaxation governed by same

relaxation constants

Two Component Viscoelastic Model

E1 1E2

2Fixed strain 0

Voigt element Maxwell element

Transverse Fibril Mechanics?

• Same two-stage

relaxation

• Fits same time constants

• Increase greater than

volume conservation alone

Relaxation Mechanics?

Fibrils

Fibres

ShorterSlide

AXIALTRANSVERSE

Increases

Relaxation Behaviour

•Significant structural reordering during relaxation

• Significant movement of water

• Some water moves out of sample?

• Water moves into fibrils?

• Transfer from fibre to fibril space?

• Each level of fibre composite independent

• Fibril response very ordered

• Fibre response opposes this

Acknowledgements

• Shima Toorani

• Vinton Cheng

• Mike Kayser

• Jong Seto

• Steffi Krauss

• Prof Steve Greenwald• Prof Julia Shelton• Prof Dan Bader• Prof David Lee

• EPSRC• Tissue Science

Laboratories

• Dr Sam Evans

• Dr Himadri Gupta