The Extracellular Matrix

Lecture 4

Objectives
! Understanding of the constituents of the ECM and the role it plays in physiological conditions. ! What do we need to know for tissue engineering. ! Understanding the types of extracellular matrices and unique characteristics for each. ! The challenge of mimicking nature. ! Knowledge on how cells interact with the matrix for signaling. ! Can we signal them artificially?

ECM: Menu Summary

! Glycosaminoglycans (GAGs)
! Packing materials, ground substances
! Cartilages

! Collagen
! Insoluble, high tensile strength
! Tendons, ligaments, cartilage, cornea

! Elastin
! Elastic
! vascular wall, skin, lung

! Fibronectin
! Cell adhesion

! Laminin
! Cell adhesion

Extracellular Matrix (ECM)

! Tissues are not made up of solely of cells. ! A substantial volume of their space is extracellular and filled by an intricate network of macromolecules called ECM. ! ECM is the non-cellular material present between cells throughout the body.

Roles of ECM ! Provide structural support. ! Provide substrates for cell adhesion. ! Regulates cellular differentiation and metabolic function.
! The ECM is composed of a variety of polysaccharides and fibrous proteins.

Types of Tissues
! Epithelial tissue: Cells are tightly attached together to form sheets (epithelia).
! ECM is scanty and forms the basal lamina.

! Connective tissues
! The matrix carries the mechanical stress to which the tissue is subjected.

ECM of the Connective tissue

! Variation in the relative amounts of the different types of matrix macromolecules and the way they are organized give an amazing diversity of forms adapting the requirement of the tissue. ! Examples. ! The ECM is made and oriented by the cells within it. Thus it is local macromolecular synthesis. ! The cells also pattern the matrix. ! ECM is mainly secreted by fibroblasts but other cells in the family can also make it. Can you think these cells?

The two main classes of ECM ! Polysaccharide chains of the class called Glycosaminoglycans (GAGs) often found covalently linked to protein (i.e. proteoglycans).

! Proteins ! Structural (collagen and elastin) fibrous protein ! Adhesive proteins (fibronectin and laminin)

Glycosaminoglycans
! ! They are unbranched polysaccharides with repeating disaccharide units. They resist compression and fill space. Why?

ECM: GAGs ! They are called GAGs because one or two sugar residues in the repeating unit are an amino sugar (N-acetylglucosamine, N-acetylgalactosamine) which are sulfated. ! The other sugar is uronic acid (Glucuronic acid, iduronic acid). ! The consequences of sulfate and carboxylate combinations is a high overall negative charge.

!Given the diversitywhat else can you think?

ECM: GAGs Summary

! Glycosaminoglycans (GAGs)
! ! ! ! ! ! Negatively charged Extended conformation Attract cations Water is sucked to the matrix Difficult to compress Packing materials, ground substances
! Cartilages, cornea

ECM: Summary
! Glycosaminoglycans (GAGs)
! Packing materials, ground substances
! Cartilages, cornea

! Collagen
! Insoluble, high tensile strength
! Tendons, ligaments, cartilage, cornea

! Elastin
! Insoluble elastic protein
! vascular wall, skin, lung

! Fibronectin and Laminin

! Cell adhesion

ECM: Fibrous Protein

! Collagen, Elastin, Fibronectin and Laminin. ! Collagen is a structural connective tissue accounting 30% of total body protein. ! (Gly-X-Y) is the primary amino acid sequence, whereby X is often proline. It also has two rare amino acids. ! 1000 amino acid residue per chain ! Many types of collagen super-family exist. Types I-III are fiber forming while IV is membrane type. ! Synthesized by fibroblasts, osteoblasts and condrocytes.

Primary amino acid sequence of collagen

-Gly-Pro-Met-Gly-Pro-Ser-Gly-Pro-Arg-Gly-Leu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp-Gly-Pro-Gln-Gly-Phe-Gln-Gly-Pro-Hyp-Gly-Glu-Hyp-Gly-Glu-Hyp-Gly-Ala-Ser-Gly-Pro-Met-Gly-Pro-Arg-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Lys-Asn-Gly-Asp-

Collagen Biosynthesis Transcription Translation Post-transnational modification

Hydroxylation Glycosylation

Procollagen assembly (triple helix) Enzymatic action Collagen fiber Fibril formation

Crosslinking methods of collagen

Collagen as scaffold for tissue engineering What we wanted to do.
!Extracellular crosslinking !aldol condensation !hydroxypyridinium !Physical crosslinking !ultraviolet (UV) ! dehydrothermal (interchain) !Chemical crosslinking !aldehyde (FA, GA) !epoxy !isocyanate !carbodiimide/NHS ! azide !others least important.

Extracellular crosslinking
Deamination

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Properties of Collagen Fiber

! In contrast to GAGs that resist compression, collagen fibrils resist tensile forces. ! Cells pattern ECM specially collagen.The cells can crawl over it and tug on it helping to compact into sheets and draw it out into cables.
! When fibroblasts are cultured on collagen gel, cells tug on it eventually forming either a fiber or shrinking it from its original size (mechanical role).

! Note the challenge we face in synthetic gels.

Elastin
! Hydrophobic protein of 750 amino acid residue. ! Contain proline and glycine like collagen but the protein is not glycosylated and has only very small hydroxylation. It is secreted in the ribosome and crosslinked at the cell surface. ! Gives tissues (bladder, skin, lung blood vessels) their elasticity as they need to be strong to function in a dynamic environment. It gives the required resilience so that they can recoil after the stretch force is released. ! Elastin fibers can stretch 5 times more than rubber bands at the same cross-sectional area. ! Large inelastic collagen fibrils are interwoven to limit the stretching and prevent the tissue from tearing.

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Courtesy of M. L. Raghavan, University of Iowa

Courtesy of M. L. Raghavan, University of Iowa

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Courtesy of M. L. Raghavan, University of Iowa

Courtesy of M. L. Raghavan, University of Iowa

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! Glycosaminoglycans (GAGs)
! Cartilages, cornea

ECM: Summary

! Packing materials, ground substances

! Collagen
! Insoluble, high tensile strength
! Tendons, ligaments, cartilage, cornea

! Elastin
! Insoluble elastic protein
! vascular wall, skin, lung

! Fibronectin and Laminin

! Cell adhesion

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