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 2

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 4

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??

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

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

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

Tissue Composition & Tissue Mechanics Articular cartilage Skin Tendon / ligament 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 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, 109-19

Considered simple collagen tissue to study Tendon Structure Tendon Fascicle Endotendon Tenocyte Fibre Crimp waveform Fibril Crimping Microfibril Tropocollagen 1.5 3.5 50-500 10-50 50-400 500-2000 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

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

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

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

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; 335-343 Scott & Thomlinson (1998) J. Anat. 192; 391-405 Screen et al (2005) Ann Biomed Eng 33; 1090-1099

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

Confocal Images – Stress Relaxation

Confocal Images – Stress Relaxation 18

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

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

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) Fibrous connective tissue

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 23

Finite Element Approach X displacement Y displacement y x

Displacements X displacement Y displacement x y

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

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

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 Confocal focus Tendon Fascicle Endotendon Tenocyte Fibre Crimp waveform Fibril Crimping Microfibril Tropocollagen 1.5 3.5 50-500 10-50 50-400 500-2000 nm mm X-ray synchrotron scattering Himadri Gupta (Max Plank)

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)

Fibril Strain During Relaxation Stress (MPa) Fibril strain (%) 60 50 40 30 20 10 0 100 200 300 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

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

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

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

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

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