Collagen and Collagenous Tissues Structure of collagen Biomechanics of collagenous tissues Testing soft collagenous connective tissues 2011: Combined Collagen and CollagenousTissues with Testing Connective Tissues 1 Feb 2001 This lecture could be improved by using pericardium and heart valves as examples of 2-D tissues and skin and cartilage as 3-D examples. I made a start on this restructuring and incorporated the heart slides into this lecture. Could also add some slides on crosslinking from Keith’s research And add a list of collagen/connective tissue diseases Perhaps also something about collagen metabolism in pregnancy.

Collagen: Overview Collagen is the primary structural protein in the body Collagen is the most prevalent protein comprising ~30% of all proteins Collagen is highly conserved between species (i.e. not undergone many evolutionary changes) Molecules arranged in staggered pattern X-ray diffraction or electron microscopy give rise to a banded pattern Also relatively resistant to enzymatic breakdown Collagen fibrillar structure

Collagen Molecular Structure Triple Helix (Gly-X-Y)N X=proline Y=hydroxyproline Triple helix + crosslinks: Structure give rise to a material that is very stiff and stable Crosslinks (covalent bonds) occur between the ends (insert diagrams) of molecules

Collagen: Molecular Biology >20 different collagen types have been identified characterized by different α-chains each coded by a different gene exons are often 54 bp long 3 bp in a codon 18 amino acids 6 sets of Gly-X-Y Homotrimer Heterotrimer Type III=(a1(III))3 Type I=(a1(I))2a2(I) Type XI=a1(XI)a2(XI)a3(XI)

Collagen Types Classifications Examples Fibrillar I Tendon, Skin, Ligament II Cartilage III Skin Vessels, Tendon V Fetal Membranes - Assoc w/ Type I VI Cartilage - Assoc w/ Type II Fibril Associated IX Cartilage, Cornea XII Embryonic Tendon XIV Fetal Skin & Tendon Network Forming IV Basement Membrane X Hypertrophic Cartilage VIII Descemets Membrane Filamentous VI Vessels, Skin Anchoring VII Anchoring Filaments Fibrillar Collagen (I (mostly), III) has greatest stiffness

Material Properties Material Stiffness UTS Collagen 1000 MPa 100 MPa Steel 200 GPa 1000 MPa Wood 10GPa 100MPa Rubber 1000-1400 kPa Bone 18000 MPa 125 MPa Elastin 500-600 kPa 100-500 MPa Silk 10000 MPa But that's not enough information to predict behavior in tissues... Tissues are composites Complex organization Complex boundary conditions

Ligaments and Tendon connect bones together (Ligament) connect bones to muscle (Tendon) Transmit forces Aid in stabilizing joint motion Absorb impacts/stresses Prevent large displacements such as dislocations Primarily uniaxial (1D) loading elements

Tendon Structure

Ligament and Tendon: Mechanical Properties

Ligaments Loading Fibers are parallel to load axis Organization fascicular organization Unloaded = crimped loaded = straight Composition Collagen 75-80% Elastin ‹5 % Proteoglycans 1-2% http://drlowe.schipul.net

Knee Ligament Structure Loaded MCL ACL Unloaded

Age Stress, MPa Strain, % Similar changes occur in collagenous tissues among individuals and most species. Progressive increases in collagen which eventually becomes more organized and cross-linked until skeletal maturity. This results in increased elastic stiffness and strength. After skeletal maturity, properties begin to deteriorate.

Advancing Age As a person becomes older, the maximal force their ACL can tolerate decreases, this is has as much to do with changes in geometry as it does changes in material properties

Immobilization Immobilization of the knee causes deterioration of the MCL material properties, but not the ACL material properties. MCL is metabolically more active, so as it remodels the tissue it lays down mechanically compromised material. However, the ACL cannot produce new tissue, so it simply atrophies.

Skin Collagen: 65 - 70 % (more type III than ligament) Elastin: 5 - 10 % Proteoglycan: 1.5 - 2 % collagen crimp decreases with age; stiffness increases elastin crimp increases with age; decreasing recoil A mechanical explanation for wrinkles? Young Adult Old

Skin: Mechanical Properties More compliant than ligament or tendon; needs to be for its functions. orientation of coiled fibers change with load collagen is stiffer that elastin but has greater hysteresis (absorbs more energy)

Ligament Tensile Testing computer Cross correlation Strain computation

Connective Tissue Testing Structural Properties describe the behavior of the actual tissue (e.g bone ligament bone complex) Mechanical properties describe the behavior of the tissue as a general material

Clamping considerations Device to hold tissue and clamping must be stiffer and stronger than the subject material. Otherwise the stiffness of the device contributes to what you measure. Ligaments—have their own "built in" clamps -- bones. Usually drill holes in bone use steel rods. Do not want to clamp too far away or elongation may include bone deformation. Do not want it too close because may damage insertion (attachment) of ligament to bone. May weaken bone. Tendons only have 1 "natural" clamp Wherever you clamp, have to worry about inhomogeneities and edge effects.

Measurement of strain Deformation of biological tissues is nonhomogeneous, i.e. the different regions can deform differently. If we use the "clamp to clamp" strain the measurements would be average over the whole region any slippage in the clamping system would also affect the measurement. Some approaches to measuring regional strain in tissues Imaging Ultrasound Strain gauges--invasive

Strain Gauges Sonomicrometry (piezoelectric crystals) www.sonometrics.com Mercury-in-rubber F Hg F V Semiconductor (resistive, peizoresistive) www.omego.com

Strain Tensors: 1D example Cauchy (infinitesimal) Lagrangian Eulerian

Stress-free state How can we identify the best stress-free 'reference' state for the stress and strain calculations? The soft tissues buckle under compression Long toe region makes it difficult to identify transition from compressive to tensile forces Solution: Use a small tare load to repeatably identify the initial state

Anelastic Properties Hysteresis Loading & unloading curves are different Area between curves represents energy absorbed by material Preconditioning Apparent material properties are history dependent Becomes repeatable with multiple cycles (in ligaments and tendons tested in vitro, this occurs between 4-7 cycles)

Viscoelastic properties Stress-Relaxation stress decreases with time but reaches an equilibrium for a step increase in strain Creep Strain gradually increases with time but reaches an equilibrium for a step load Strain-Rate Effects increased strain rate results in increased stiffness due to viscous forces These effects are small in ligaments and tendons for the normal range of strain rates, but can be important in relation to prevention of injury

6 DOF Knee Testing Rig

Collagenous Tissues: Key Points Collagen is a ubiquitous structural protein with many types all having a triple helix structure that is cross-linked in a staggered array. Some of the most common collagen types are fibrillar and the collagen can be organized in 1-D, 2-D or 3-D in different tissues to confer different material properties. The 1-D hierarchical arrangement of stiff collagen fibers in ligaments and tendons gives these tissues high tensile stiffness The 2-D arrangement of collagen fibers in tissues such as skin is often quite wavy or disordered to permit higher strains Crimping, coiling and waviness of collagen matrix gives the tissue nonlinear properties in tension. Collagen structure in tissues changes with disease & ageing. Different tissue types require different testing configurations.