Tissue Engineering & Drug Delivery BBI 4203 Lecture 4 Extracellular Matrix (ECM)

Cell-cell and cell-matrix interaction

Roles of extracellular matrix (ECM) Structural support - tensile strength, stiffness, elasticity Cell adhesion - many cells die in suspension - they must adhere to a surface Cell morphology Cell proliferation/differentiation and metabolism - second messenger action (focal adhesion kinases) Cell migration - neurons trying to connect up with all the proper muscles - embryogenesis

Extracellular matrix - composition Three-dimensional array of protein fibers embedded in the hydrogel made of glycosaminoglycans (GAGs) Primary ECM components Polysaccharide chains (carbohydrates) - Glycosaminoglycans (GAGs) - Proteoglycans Fibrous proteins - Collagen - Elastin - Fibronectin - Laminin Structural Adhesive

Polysaccharides Condensation polymers made from sugar monomers Primarily modified glucose Water absorbing or water wicking Structural polysaccharides Cellulose, chitin Energy storing polysaccharides Starch and glycogen Lubrication and viscous damping Ground substance

Simple and complex carbohydrates

Simple sugars

Polysaccharides

Cellulose

Ground substance Gel-like matrix embedded with proteins and cells Viscous aggregate of water-swollen polysaccharides linked to proteins Acts as shock absorber and lubricant Glycosoaminoglycans Hylanuronic acid backbone Proteoglycan monomers Link proteins Hylauronic acid gel

Amorphous gel matrix interspersed between cells, collagen and elastin A=fibroblasts, B=collagen fibers, C=elastin fibers, D=reticular fibers

Proteoglycan aggregate: “bottle brush” structure of ground substance Backbone High mwt and linear GAG of HA Proteoglycan monomer “bristles” Low mwt linear GAG linked to core protein Link protein links monomer to HA backbone Higher density of bristles the more hydrated the tissue

Glycosoaminoglycans (GAGs) GAG: net negatively charged and polar strands get heavily solvated, which interacts strongly w/ positive charges and H-bonds w/ water Hyaluronic acid (HA) is main component of GAG Linear polysaccharide of copolymer D-gluconate and N-acetyl-D-glucosamine 1000kDa Smaller GAGs make up bristles Chondroitin-4-sufate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate 10-35kDa

Glycosaminoglycans (GAGs) Unbranched polysaccharide chains composed of repeating disaccharide units Relatively inflexible, water soluble, coil in random configuration 10% in weight Occupy a large amount of space and form hydrated gels Negatively charged since sulfated (S03-) Main groups: Hyaluronan (hyaluronic acid) Dermatan sulfate Chondroitin sulfate Heparin sulfate Heparin Keratin sulfate Typically 70-200 sugars long

Types of GAGs

Hyaluronan (hyaluronic acid) Unsulfated, not attached to protein, free of other sugar groups Facilitates cell migration Other GAGs attached to protein – they form Proteoglycans (up to 95% sugar)

Proteoglycans Cartilage, synovial fluid

Tissue hydration Hydrated tissues: 80-100 proteoglycan brushes attached per HA molecule Connective tissue and cartilage Low hydrated tissues: 1-2 proteoglycan brushes per HA molecule Cornea Nonhydrated tissues: no ground substance Hair and nails

Hyaline cartilage

Collagen: primary structural protein in the body 12/19/2017 Collagen: primary structural protein in the body Highly redundant, highly oriented structure -- “rope”

Collagen primary structure 12/19/2017 Collagen primary structure Five types of collagen Types I, II, III, IV, V All with the same basic structure [-Gly-Pro-Hyp-Gly-X-]n ~31-34% ~12-14% ~9-10% X=Aspartic acid, Glutamic acid, Asparagine, Lysine, Arginine, Histidine (all polar ==> H bonding)

Secondary structure Left-handed alpha helix Pro & Hyp bulky 12/19/2017 Secondary structure Left-handed alpha helix Pro & Hyp bulky Force helical structure Nature’s polypropylene

Tertiary structure Right-handed triple helix Coiled-coil Tropocollagen 12/19/2017 Tertiary structure Right-handed triple helix Coiled-coil Tropocollagen Trimeric collagen molecule

Quaternary structure Collagen molecules pack into fibrils 12/19/2017 Quaternary structure Collagen molecules pack into fibrils Banded appearance due to density of fibril overlap

Crosslinking of tropocollagen to form collagen fibrils 12/19/2017 Crosslinking of tropocollagen to form collagen fibrils

Laminin Large heterotrimeric cross-shaped protein

Laminin structure Three polypeptide subunits a (5 types), b (3), and g (3) At least 12 different types found in mammals - Laminin 1 consists of a1, b1, g1 - Laminin 5 consists of a3A, b3, g2 Binding domains for: - Collagen IV - Heparin - Heparan sulfate - Cell binding (integrin) - Cell specific binding (nerve, liver)

Laminin function

Basal lamina - proteins (entactin)

Collagen and Elastin

Skin, ligament and tendon collagen elastin ligament tendon skin stress strain

Specialized connective tissue Un-mineralized connective tissue Loose and dense irregular connective tissue Tendon Ligament Cartilage Blood vessels Skin Mineralized connective tissue Bone Teeth