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 factors
Nutrients
Mechanical
cues:
Fluid flow
Pressure
Deformation
Mechanotransduction
Mechanical
Loading
(in vitro)
(in vivo)
•
•
•
•
Altered Cell
Response
Proliferation
Matrix synthesis
Matrix degradation
Cell/matrix orientation
Regulates normal tissue homeostasis
Implicated in pathological processes
Implicated in repair processes
Harness it for tissue engineering??
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
Tissue Composition & 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 & Tissue
Mechanics
Articular cartilage Tendon / ligament
Skin
Aortic valve
In Situ Analysis Techniques
• 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
Specimen
Medium
Stepper Motor
Grips
Coverslip
Microscope
Objective Lens
Heater Pads
Screen et al. (2003)
Screen et al. (2004) J.
Biorheol. 40, 361-8
Eng. Med. 218, 109-19
Tendon Structure
Tenocyte
Fibre
Fibril
Fascicle
Tendon
Tropocollagen
Endotendon
Microfibril
Crimping
1.5
3.5
50-500
Crimp
waveform
10-50
nm
Multi-level fibre composite
50-400
mm
500-2000
Considered simple
collagen tissue to study
Tendon Extension Mechanisms
Fibre Sliding
Fibre Extension
u
Fibre Sliding
Fibre Extension
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
u
Screen
0 et al.2 (2004)4 J. Strain
6 40:4,
8 157-163
Applied Strain (%)
Between group displacement
(% applied displacement)
Within-group strain (%)
v
L
5
4
3
v
2
L
1
0
0
2
4
6
Applied Strain (%)
8
Tendon Extension Mechanisms
Collagen molecule
Fibril
Fibre
Fascicle
Tendon Extension Mechanisms
Extension
Collagen molecule
Fibril
Shearing/
Sliding
Fibre
rotation
Fascicle
What controls the fibre composite
behaviour?
• Non-Collagenous Matrix
• Decorin: Binds around collagen fibrils
Shape Molecule
Scott & Thomlinson
J. Anat.
192; 391-405
Screen(1998)
et al (2005)
Ann Biomed
Eng 33; 1090-1099
Scott (2003)
J. Physiol. 553; 335-343
Understanding Viscoelasticity
Gross mechanical properties:
Direct tests
2.5
Incremental tests
2.5
8%
2
Force (N)
2
Force (N)
6%
1.5
1
1.5
0.5
0
0
100
200
300
400
Time (secs)
500
4%
1
0.5
0
8%
2%
0
100
200 300
Time (secs)
• Very rapid relaxation ; Total relaxation < 60 secs
• Highly viscous tissue
400
500
Confocal Images – Stress Relaxation
Confocal Images – Stress Relaxation
Confocal Images – Stress Relaxation
Applied Extension = L
Fibre Relaxation
tenocyte
nuclei
collagen
fibre
Fibre Siding
Confocal Images – Stress Relaxation
TYPICAL DATA:
4 % Applied Strain
Fibre Sliding
Percentage fibre relaxation (%)
0.5
0.4
y = -0.0002x + 0.0151
2
0.3
R = 0.0034
0.2
0.1
0
-0.1 0
20
-0.2
-0.3
-0.4
-0.5
Time (secs)
40
60
Percentage between-fibre relaxation (%)
Fibre Relaxation
0.5
0.4
y = -0.0029x - 0.214
0.3
2
R = 0.6649
0.2
0.1
0
-0.1 0
20
40
-0.2
-0.3
-0.4
-0.5
Time (secs)
60
Confocal Images – Stress Relaxation
Fibre Relaxation
1%
2%
4%
6%
Fibre relaxation (mm)
0.01
0
-0.01
-0.02
-0.03
-0.04
-0.05
-0.06
Applied Strain (%)
1%
8%
Between-fibre displacement (mm)
0.02
Fibre Sliding
2%
4%
6%
0
-0.5
-1
-1.5
-2
-2.5
Applied Strain (%)
8%
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
Important coordinates into Matlab
Construct a Delaunay mesh of
triangle elements
Monitor deformation & strain in
each element during relaxation
S Evans - Cardiff University
Finite Element Approach
X displacement
y
x
Y displacement
Displacements
X displacement
y
x
Y displacement
Relaxation Strains
X strain
Y strain
Shear strain
y
x
Huge variability in response
Strain seems random
Relaxation Strains
25
25
y strains
x strains
20
20
15
15
10
10
5
Predominantly negative
= compression
Range positive & negative
= Fibre sliding
5
0
0
-0.4 -0.3 -0.2 -0.2 -0.1
0
0.08 0.16 0.24 0.32 0.4
-0.4 -0.3 -0.2 -0.2 -0.1
Strain
Strain
25
20
shear strains
15
10
y
5
x
0
Wide range of shear strains
0
-0.4 -0.3 -0.2 -0.2 -0.1
0
0.08 0.16 0.24 0.32 0.4
Shear strain
0.08 0.16 0.24 0.32 0.4
Relaxation Behaviour
Loading Direction:
• 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)
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 Changes
Tenocyte
Fibril
Fibre
Fascicle
Tendon
Tropocollagen
Endotendon
Microfibril
Crimping
1.5
3.5
50-500
Crimp
waveform
10-50
nm
mm
50-400
500-2000
Confocal focus
X-ray synchrotron scattering
Himadri Gupta (Max Plank)
Synchrotron X-ray Scattering
Small angle X – ray
scattering (SAXS) setup
ESRF BL ID2
Peter Boesecke
(Grenoble)
CCD X – ray
detector
Load cell
2/D
X - ray
Microtensile tester
Max load 250 g – 12 kg
Strain measured with video extensometry
(NON-contact)
Fibril Strain During Relaxation
Time (Seconds)
0
0
100
100
200
200
300
300
60
data
fit
fit, ef tcs
20
stress [MPa]
Stress (MPa)
50
40
18
30
 t 
 t 
ε F (t)  ε F 1 exp     ε F 2 exp     ε F 0
 τ 
 τ 
16
20
10
 t 
 t 
σ(t)  Δσ 1 exp     Δσ 2 exp     σ 0
 τ 
 τ 
14
0
1.58
(%)
Fibril
fibril strain
strain [%]
Two time constants + , -
2.5
1.56
General Form
1.54
2.0
1.52
1.50
1.5
1.48
1.46
1.0
1.44
1.42
0
0
100
100
200
200
Time (Seconds)
300
300
Fitting ‘ε’ constants to ‘σ’ ?
Fitting Data:
Fibrilσ relaxation
& stress
ε
+ & + ≤ 10 s
relaxation
governed
by same
σ
ε
- & - ≥ 50 s
relaxation constants
Two Component Viscoelastic Model
2
Fixed strain 0
E1
1
E2
Voigt element
Maxwell element
Transverse Fibril Mechanics?
• Same two-stage
relaxation
• Fits same time constants
• Increase greater than
volume conservation alone
Relaxation Mechanics?
TRANSVERSE
AXIAL
Shorter
Slide
Increases
Fibres
Fibrils
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
• Dr Sam Evans
• Mike Kayser
• Dr Himadri Gupta
• Jong Seto
• Steffi Krauss
•
•
•
•
Prof Steve Greenwald
Prof Julia Shelton
Prof Dan Bader
Prof David Lee
• EPSRC
• Tissue Science
Laboratories