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Collagen and Collagenous Tissues • Structure of collagen • Biomechanics of collagenous tissues • Testing soft collagenous connective tissues 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 Band Spacing D=670 Å FIBRIL Hole Zone (0.6D) Overlap Zone (0.4D) MICROFIBRIL 3000Å (4.4D) COLLAGEN MOLECULE 15Å Dia 104Å TRIPLE HELIX PRIMARY STRUCTURE IN a-CHAIN 8.7Å Glycine Y X Glycine Y X Collagen: Molecular Biology • >20 different collagen types have been identified • characterized by different -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 Type III=(a1(III))3 Heterotrimer Type I=(a1(I))2a2(I) Type XI=a1(XI)a2(XI)a3(XI) Collagen Types Classifications Fibrillar I II III V VI Fibril Associated IX XII XIV Network Forming IV X VIII Filamentous VI Anchoring VII Examples Tendon, Skin, Ligament Cartilage Skin Vessels, Tendon Fetal Membranes - Assoc w/ Type I Cartilage - Assoc w/ Type II Cartilage, Cornea Embryonic Tendon Fetal Skin & Tendon Basement Membrane Hypertrophic Cartilage Descemets Membrane Vessels, Skin Anchoring Filaments Fibrillar Collagen (I (mostly), III) has greatest stiffness Material Properties Material Collagen Steel Wood Rubber Bone Elastin Silk Stiffness 1000 MPa 200 GPa 10GPa 1000-1400 kPa 18000 MPa 500-600 kPa 10000 MPa UTS 100 MPa 1000 MPa 100MPa 125 MPa 100-500 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 100 Tensile Strain Tensile Stress Stress (MPa) 75 50 ge n Ta 25 0 0 2 M t n lu u od 4 Toe Region Linear Region 6 s 8 Yielding and Microfailures Strain (%) 10 Catastrophic Failure 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 Lateral Femur Medial Quadriceps Tendon Patella Lateral Collateral Ligament Menisci (LCL) Medial Collateral Ligament (MCL) Posterior Cruciate Ligament (PCL) Fibula Anterior Cruciate Ligament (ACL) Tibia Patellar Tendon Knee Ligament Structure Loaded MCL ACL Unloaded • • • • 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. Stress, MPa Age Strain, % 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) Mercury-in-rubber www.sonometrics.com F Hg F V Semiconductor (resistive, peizoresistive) www.omego.com Strain Tensors: 1D example Cauchy (infinitesimal) L L 11 1 1 L0 L0 Lagrangian 1 2 1 L2 L20 E11 1 2 2 L20 Eulerian 1 1 1 L2 L20 e11 1 2 2 2 L2 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.