1. Hierarchical Approaches to Investigating Tissue Micromechanics
Hazel Screen
School of Engineering & Materials Science,
Queen Mary, University of London
6th November 2008

2. 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 2

3. Investigating Tissue Micromechanics
1. Understanding tissue structure and how to help protect it from damage
2. Understand how to facilitate repair in damaged tissue

4. How to Facilitate Tissue Repair
Chemical cues:
Growth factors
Nutrients
Mechanical cues:
Fluid flow
Pressure
Deformation 4

5. Cell/matrix orientation
Mechanotransduction
Altered Cell Response
Mechanical
Loading
(in vitro)
(in vivo)
Proliferation
Matrix synthesis
Matrix degradation
Cell/matrix orientation
It is thought that mechanical stimuli are detected by the tenocytes, initiating mechanostransduction pathways, which can result in ……
These mechanostranduction pathways are important for regulating normal tissue homeostatis (as seen from the matrix response to stress deprivation) but also have a role in pathological and repair processes
If these pathways can be interpreted, they can also be utilised to upregulate desired matrix components in tissue engineered constructs.
Regulates normal tissue homeostasis
Implicated in pathological processes
Implicated in repair processes
Harness it for tissue engineering??

6. The Hierarchy of Mechanobiology
Tissue mechanics
Cell mechanics
Protein mechanics
Joint mechanics
Body mechanics 6

7. The Hierarchy of Mechanobiology 
Tissue mechanics
Cell mechanics
Protein mechanics
Joint mechanics
Body mechanics 7

8. 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 8

9. Tissue Composition & Tissue Mechanics
Articular cartilage
Skin
Tendon / ligament
Aortic valve

10. 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
Stepper Motor
Heater Pads
Microscope
Objective Lens
Grips
Specimen
Medium
Coverslip
So, another mechanisms of examining the micromechanics of the tissue and establishing the effects of micromechanics on mechanotransduction is to look at the cell response using confocal microscopy, and we have produced a number of papers outline this methodology, whereby it is possible to look at the cells themselves and see how they respond, and also to use the cells as markers of the strain fields within the tissue
Screen et al. (2003) Biorheol. 40, 361-8
Screen et al. (2004) J. Eng. Med. 218,

11. Considered simple collagen tissue to study
Tendon Structure
Tendon
Fascicle
Endotendon
Tenocyte
Fibre
Crimp waveform
Fibril
Crimping
Microfibril
Tropocollagen nm mm
So, if we first assess what we know about the structure,
We know tendon is a fibrous connective tissue with a hierarchical arrangement of collagen as shown above.
Considered simple collagen tissue to study
Multi-level fibre composite

12. Tendon Extension Mechanismsu
Fibre Extension
Fibre Sliding v L u
Fibre Extension v L
Fibre Sliding
Screen et al. (2004) J. Strain 40:4,
So taking a series of cell nuclei, we can use these as cell nuclei to examine whether tendon extension occurs predominantly as extension of a fibre, or sliding between fibres. And we can see from the mean data curves here the fibre extension tends to level off after the toe region, and further tendon extension relies on sliding between the fibres

13. Tendon Extension Mechanisms
Collagen molecule
Fibril
Fibre
Fascicle
SO IF WE CONSIDER OUR TENDON HIERARCHY:
The matrix enables extension by extension of the individual collagen molecules, but also varying degrees of sliding throughout the tendon hierarchy
But this then also tells us that the cells, located here between fibres must respond to mechanotransduction cues that are predominantly shear in nature, hence effects such as deformation of the long and complex cell processes may be of particular importance for tenocyte mechanotransduction
Different types or levels of deformation alter the mechanotranduction response

14. Tendon Extension Mechanisms
Collagen molecule
Fibril
Fibre
Fascicle
Shearing/
Sliding
SO IF WE CONSIDER OUR TENDON HIERARCHY:
The matrix enables extension by extension of the individual collagen molecules, but also varying degrees of sliding throughout the tendon hierarchy
But this then also tells us that the cells, located here between fibres must respond to mechanotransduction cues that are predominantly shear in nature, hence effects such as deformation of the long and complex cell processes may be of particular importance for tenocyte mechanotransduction
Different types or levels of deformation alter the mechanotranduction response
rotation

15. What controls the fibre composite behaviour?
Non-Collagenous Matrix
Decorin: Binds around collagen fibrils
Shape Molecule
Coming at this from a very different angle, a number of electron microscopy studies have looked at the non-collagenous matrix between the collagen fibrils, and observed the presence of GAG chains spanning the space between the fibrils. The real pioneer of this work has been Scott, and this image is from his 1998 paper. In light of these findings, he has developed a shape molecule theory, showing how PG core protein attach to collgen fibrils, and the GAG chains could interlink adjacent fibrils.
Scott (2003) J. Physiol. 553;
Scott & Thomlinson (1998) J. Anat. 192;
Screen et al (2005) Ann Biomed Eng 33;

16. Understanding Viscoelasticity
Gross mechanical properties:
Direct tests
Incremental tests 8% 8% 6% 4% 2%
Fibrous connective tissue
Very rapid relaxation ; Total relaxation < 60 secs
Highly viscous tissue

17. Confocal Images – Stress Relaxation

18. Confocal Images – Stress Relaxation18

19. Confocal Images – Stress Relaxation
Applied Extension = L
Fibre Relaxation
tenocyte nuclei
Fibrous connective tissue
collagen fibre
Fibre Siding

20. Confocal Images – Stress Relaxation
TYPICAL DATA:
4 % Applied Strain
Fibre Relaxation
Fibre Sliding
Percentage fibre relaxation (%)
Percentage between-fibre relaxation (%)
Fibrous connective tissue

21. Confocal Images – Stress Relaxation
Fibre Relaxation
Fibre Sliding
1% 2% 4% % 8%
1% 2% 4% 6% 8%
Fibre relaxation (mm)
Between-fibre displacement (mm)
Fibrous connective tissue

22. 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?

23. 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 23

24. Finite Element Approach
X displacement
Y displacement y x

25. Displacements
X displacement
Y displacement x y

26. Relaxation Strains x Huge variability in response Strain seems random
X strain
Y strain
Shear strain x y
Huge variability in response
Strain seems random

27. Range positive & negative Predominantly negative
Relaxation Strains
y strains
x strains
Range positive & negative
= Fibre sliding
Predominantly negative
= compression
shear strains x y
Wide range of shear strains

28. 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

29. Cell Perspective Cell processes link adjacent rows of cells:
Large deflections (y strains)
Compressive loading of cells (x strains)

30. Other Hierarchical Changes
Confocal focus
Tendon
Fascicle
Endotendon
Tenocyte
Fibre
Crimp waveform
Fibril
Crimping
Microfibril
Tropocollagen nm mm
X-ray synchrotron scattering Himadri Gupta (Max Plank)

31. Synchrotron X-ray Scattering
ESRF BL ID2
Peter Boesecke (Grenoble)
CCD X – ray detector
X - ray
Load cell
Small angle X – ray scattering (SAXS) setup
Microtensile tester
Max load 250 g – 12 kg
2/D
Now looking at the fibril level, we used small angle synchrotron x-ray diffraction to monitor the properties of the fibril during relaxation. Samples were loaded into the rig shown schematically on the right, and placed into the x-ray beam, using a small angle set up to provide data on fibril strain, by monitoring the D period spacing between collagen modules.
Here we can see a photograph of the set up, with a sample ready for testing, and on the left is a diffraction pattern recorded from one of the samples, showing the characteristic axial diffraction pattern created by the ordered stagger of collagen molecules. By looking at movement of the peaks, as indicated here, a measurement of fibril strain can be achieved.
Strain measured with video extensometry
(NON-contact)

32. Fibril Strain During Relaxation
Stress (MPa)
Fibril strain (%) 60 50 40 30 20 10
Time (Seconds)
2.5
2.0
1.5
1.0
Two time constants + , -
Fitting Data:
+σ & +ε ≤ 10 s
-σ & -ε ≥ 50 s
General Form
So taking this data, we are now trying to develop a model to help describe the response, and understand what is happening.
Starting from our most typical form, the rapid relaxation followed by a slower decay indicates that there are two time constants, and we can create general equations to describe the relaxation: both stress relaxation and fibril relaxation.
Looking at the time constants required to fit these data, we see that T+ for both the stress and the fibril must be less than 10 seconds, while T- for both the stress and the fibril must be greater than 50 seconds.
Moving on from here, if can try to fit the fibril strain relaxation constants to the gross stress relaxation to assess the fit, we see the following, where the solid line is the fit using the original stress constants and the dotted line the fit using the fibril strain constants.
You can see how close these are, indicating the fibril relaxation and the gross sample stress relaxation are governed by the same relaxation constants.
Fitting ‘ε’ constants to ‘σ’ ?
Fibril relaxation & stress relaxation governed by same relaxation constants 32

33. Two Component Viscoelastic Model1 E2 2
Fixed strain 0
Voigt element
Maxwell element
So what does this mean.
We are the process of building a a model to describe our data, using a Voight element and a Maxwell element.
Our Voight element can describe our collagen components within the system, while the Maxwell element describe the proteoglycans, so we’re looking at the stiff not particularly viscous collagen and the significantly more viscous and ductile PGs. Using this model, we can start to develop a relationship between the two components, to help describe how the viscous proteoglycan matrix may enable the non zero strain rates…work in progress. 33

34. Transverse Fibril Mechanics?
Same two-stage relaxation
Fits same time constants
Increase greater than volume conservation alone

35. Relaxation Mechanics? TRANSVERSE AXIAL Fibres Fibrils Shorter
Increases
AXIAL
Shorter
Slide 35

36. Relaxation Behaviour Each level of fibre composite independent
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

37. Acknowledgements Shima Toorani Vinton Cheng Mike Kayser Jong Seto
Steffi Krauss
Dr Sam Evans
Dr Himadri Gupta
Prof Steve Greenwald
Prof Julia Shelton
Prof Dan Bader
Prof David Lee
EPSRC
Tissue Science Laboratories