Hierarchical Approaches to Investigating Tissue Micromechanics Hazel Screen School of Engineering & Materials Science, Queen Mary, University of London.
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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
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