Hierarchical Approaches to Investigating Tissue Micromechanics

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

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

5. Mechanotransduction Altered Cell Response Mechanical Loading (in vitro) (in vivo) 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??

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

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

8. Investigate the local mechanical environment as the mechanotransduction stimulus of interest Tissue Composition & Mechanics • How does the tissue hierarchy control mechanical properties? • How does the material deform: • How are strains transferred to the cells?

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

10. In Situ Analysis Techniques Specimen Medium Stepper Motor Grips Heater Pads Microscope Objective Lens Coverslip • 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 Screen et al. (2003)Biorheol. 40, 361-8 Screen et al. (2004)J. Eng. Med. 218, 109-19

11. Tenocyte Tendon Fascicle Fibre Fibril Tropocollagen Endotendon Microfibril Crimp waveform Crimping 1.5 3.5 50-500 10-50 50-400 500-2000 mm nm Tendon Structure Considered simple collagen tissue to study Multi-level fibre composite

12. Tendon Extension Mechanisms Fibre Sliding Fibre Extension Fibre Sliding Fibre Extension u u v v L L Screen et al. (2004) J. Strain 40:4, 157-163

13. Tendon Extension Mechanisms Collagen molecule Fibril Fibre Fascicle

14. Extension Shearing/ Sliding rotation Tendon Extension Mechanisms Collagen molecule Fibril Fibre Fascicle

15. Decorin: Binds around collagen fibrils Shape Molecule • Non-Collagenous Matrix What controls the fibre composite behaviour? Scott & Thomlinson (1998) J. Anat. 192; 391-405 Screen et al (2005) Ann Biomed Eng 33; 1090-1099 Scott (2003) J. Physiol. 553; 335-343

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

17. Confocal Images – Stress Relaxation

18. Confocal Images – Stress Relaxation

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

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

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

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 Important coordinates into Matlab Track coordinates of every cell Construct a Delaunay mesh of triangle elements Monitor deformation & strain in each element during relaxation S Evans - Cardiff University

24. X displacement Y displacement Finite Element Approach y x

25. X displacement Y displacement y x Displacements

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

27. y x Relaxation Strains y strains x strains Range positive & negative = Fibre sliding Predominantly negative = compression shear strains 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. Tenocyte Tendon Fascicle Fibre Fibril Tropocollagen Endotendon Microfibril Crimp waveform Crimping Confocal focus 1.5 3.5 50-500 10-50 50-400 500-2000 mm nm X-ray synchrotron scattering Himadri Gupta (Max Plank) Other Hierarchical Changes

31. Synchrotron X-ray Scattering ESRF BL ID2 Peter Boesecke (Grenoble) Small angle X – ray scattering (SAXS) setup Load cell X - ray CCD X – ray detector 2/D Microtensile tester Max load 250 g – 12 kg Strain measured with video extensometry (NON-contact)

32. Fibril Strain During Relaxation Time (Seconds) 0 100 200 300 60 50 40 30 20 10 0 Stress (MPa) 2.5 2.0 1.5 1.0 Fibril strain (%) General Form Fitting Data: +σ & +ε ≤ 10 s -σ & -ε ≥ 50 s 0 100 200 300 Time (Seconds) Two time constants + , - Fitting ‘ε’ constants to ‘σ’ ? Fibril relaxation & stress relaxation governed by same relaxation constants

33. Two Component Viscoelastic Model Fixed strain 0 2 E2 E1 1 Maxwell element Voigt element

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

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

36. 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

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