Components of tissue engineering


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Components of tissue engineering

  1. 1. Tissue Engineering Overview
  2. 2. • Can I live with a beating heart that came from no one?
  3. 3. • Interdisciplinary field that applies the principle of engineering and life sciences to the development of biological substitutes that restore, maintain or augment tissue function
  4. 4. Tissue Engineering• An alternative to drug therapy, gene therapy and whole organ transplantation – Gene and drug therapy an option for treating the underlying disease if the molecular basis of the disease is understood – Less suitable for replacing the entire function of the cell – “Grow” organs in the lab
  5. 5. Regulators of Matrix Assembly InsolubleSoluble Matrix Matrix Molecules Assemblies CELLS Matrix BoundSoluble Growth Growth Factors Factors Bioactive Cells Matrix
  6. 6. Steps in Tissue Engineering• Appropriate cell source must be identified, isolated and produced in sufficient numbers• Appropriate biocompatible material that can be used as a cell substrate or cell encapsulation material isolated or synthesized, manufactured into desired shape and dimensions• Cells seeded onto or into material, maintaining function, morphology• Engineered structure placed into appropriate in vivo site
  7. 7. Extracellular Matrix• Cell growth and differentiation in 2D cell culture and 3D organ culture requires presence of structured environment with which cells can interact• ECM – polymeric networks of several types of macromolecules in combination with smaller molecules, ions and water
  8. 8. ECM• Composed of: – Fibrous proteins • Collagens • Elastin • Fibrillin • Fibronectin • Laminin – Hydrophilic proteoglycans• Assembled by cells, modified by cells as they proliferate, differentiate, and migrate
  9. 9. • Recognized that it is not inert• Influences cell shape, fate, metabolism• Detailed characterization of ECM essential for understanding behaviour of cells• Structure, signaling, regulators of cell behaviour• Hugely varied – Hard tissues of bone and teeth – Transparent matrix of the cornea – Ropelike organization of tendons
  10. 10. • GAG and proteoglycan molecules form highly hydrated gel-like “ground substance” in which the fibrous proteins are embedded• Aqueous phase permits diffusion of nutrients• Collagen fibres strengthen and organize matrix• Elastin fibres give resiliance• Adhesive proteins help cells to attach to ECM
  11. 11. • Secreted in many cases by cells as precursor molecules• Significantly modified before assembly with other components into functional polymers – Proteolytically processed – Sulfated – Oxidized – Cross linked• Formation is unidirectional, irreversible• Polymers reconstituted in lab with components extracted from ECM do not have all properties as when assembled by cells
  12. 12. • ECM is also modified by cells as they proliferate, differentiate, and migrate• Cells continually interact with matrix• Communication pathway• ECM influences cell shape, fate and metabolism• Understanding of ECM is therefore essential to understanding cell behaviour in context of tissue and organ development and function – Structural components (collagen, elastin) – Signalling molecules (matrix bound GF’s) – Multidomain molecules
  13. 13. Collagens• Major scaffold proteins of ECM• Family of proteins• Most abundant protein in mammals, up to 30% of all proteins• Responsible for functional integrity of tissues such as cartilage, skin, tendon• 15 collagen types present in human tissues• High tensile strength, equivalent to steel when compared on cross-sectional area, factor of three greater on a per weight basis
  14. 14. The Collagen MoleculeDiagram from Nimni Diagram page 49 TE book
  15. 15. The Collagen Molecule∀ α chain – Gly-X-Y tripeptide sequence – Y frequently Pro, Hyp – Proline, OH-proline follow each other relatively frequently – Gly-Pro-Hyp sequence makes up about 10% of molecule – Types I-III collagen, MW 100 kDa, 1000 amino acids – Stabilized by hydrogen bonds (1-2 per 3 amino acids – Molecular rods 30 nm in length, 1.5 nm in diameter
  16. 16. FibrillogenesisFigure 9 Nimni
  17. 17. Types of Collagen Figure 11 Nimni
  18. 18. Type I Collagen• Three chains, two α1 chains, 1 α2 chain• Abundant in skin, tendon, ligament, bone, cornea – 88-99% of total collagen
  19. 19. Type II Collagen• Present in large amounts in cartilage• Also present in intervertebral disk, vitreous humour of the eye
  20. 20. Type III Collagen• Present in small amounts in skin, larger amounts in blood vessels, absent in bone• Associated with Type I collagen• Seems to located predominantly at the fibril surface, appears to mediate interactions between fibrils, important for mechanical properties of tissues
  21. 21. Figure 12 from Nimni
  22. 22. • Other structural or fiber forming collagens – Types V and IX• Type V collagen is abundant in vascular tissues produced by blood vessels• Also present in avascular corneal stroma
  23. 23. Basement Membrane Collagens• Type IV collagen major component of basement membranes• Does not organize into fibrillar structure• Resembles procollagen with carbohydrates accounting for 10% of the mass• Associated with a large number of non- collagenous molecules as well as Type VII collagen
  24. 24. Elastin• Source of elasticity in tissues• Prominent in lung, skin and blood wall
  25. 25. Elastin• Necessary for providing tissue with elasticity so that they can recoil after transient stretch• Extensibility that is five times that of elastic band with same cross-sectional area• Highly insoluble• Composed of alternating hydrophobic and Ala and Lys rich crosslinking domains• Hydrophobic domains contain repetitive sequences of 3-9 uncharged amino acids
  26. 26. • Lys domains oxidized by enzyme lysyl oxidase to form aldehydes and extensive crosslinks between neighbouring molecules in the fibre• Elasticity driven by hydrophobic interactions, tendency of hydrophobic segments to adopt a random coil configuration following stretch• Tropoelastin – soluble precursor of elastin• Can form extensive crosslinks with multiple adjacent tropoelastins providing for potential extensive networking
  27. 27. Microfibrils• Other component of elastic fibers• Complex of glycoproteins organized into small 10-12 nm diameter tubular fibrils• Fibrillin major component• Contain many charged and basic amino acids including cysteines• Importance highlighted in diseases including Marfan syndrome
  28. 28. • Other molecules (proteoglycan) are seen in association with elastin including – Decorin – Hyaluronic acid – Dermatan sulfate• May provide hydration necessary for elastic recoil or prevent spontaneous aggregation of tropoelastin in extracellular space allowing fibrillogenesis to occur
  29. 29. Tissue Distribution of Elastic Fibres• Abundant is tissues subjected to repetitive deformation – Blood vessel wall – Alveolar septal interstices – Deep dermal layers – Elastic cartilage• Amount varies depending on physical demands on tissue – 30-75% of dry weight of tissue
  30. 30. • Organized into three distinct morphological forms – Elastic ligaments skin and lungs – fibers are small and rope-like – In blood vessels – concentric sheets or lamellae interconnected by fine elastic fibers – Cartilage – organize as trabecular network
  31. 31. Glycosaminoglycans• Long, unbranched polysaccharide chains composed of repeating sugar units• 70-200 sugar residues long• Highly negatively charged due to sulfate and carboxyl groups• One of two sugar residues in repeating disaccharide is always an amino sugar – N-acetylglucosamine – N-acetylgalactosamine
  32. 32. • Four main groups of GAGs, distinguished by sugar residues, type of linkage between residues and number and location of sulfate groups – Hyaluronic acid – Chondroitin sulfate and dermatan sulfate – Heparan sulfate and heparin – Keratan sulfate
  33. 33. • Too inflexible to fold into compact globular structures• Strongly hydrophilic• Tend to adopt highly extended random coil configurations, huge volume relative to mass• Form gels, even at very low concentrations, filling most of the extracellular space, providing mechanical support for the tissues
  34. 34. The GlycosaminoglycansGAG MW A B Sulfates Protein Other Tissues SugarsHA 4000 – Glucuronic Glucos- 0 - 0 Skin, 8x106 acid amine vitreous, cartilageCS 5000- Glucuronic Galacto 0.2 – 2.3 + Galactos Cartilage 50000 acid s-amine exylose Cornea BoneHS 5000- Glucuronic Glucos- 0.2-2.0 + Galactos Lung, 12000 acid amine exylose arteriesKS 4000- Galactose Glucos- 0.9-1.8 + Galactos- Cartilage 19000 amine amine cornea
  35. 35. Proteoglycans• Core protein with one or more covalently bound linear polysaccharide chains (GAGs)• Important in migrating and proliferating cells• Allow cartilage to withstand compressive forces• Regulate adhesion, migration, proliferation, mechanical roles
  36. 36. Proteoglycans• Except for HA, all GAG’s found linked to protein• Usually easily distinguishable from glycoproteins by nature and arrangement of sugar side chains• Glycoproteins 1-60% carbohydrate by weight, 300 000 Da or less• Proteoglycans – up to 95% carbohydrate by weight – 3 000 000 Da or more
  37. 37. • Potential for limitless heterogeneity• Can differ markedly in protein content, molecular size, number and type of GAGs• Very difficult to characterize and classify
  38. 38. Function of Proteoglycans• Bind various secreted signaling molecules in vitro• Form gels of varying pore size and charge density, functioning as sieves to regulate traffic of molecules and cells• Difficult to determine arrangement in vivo since highly water soluble and readily washed away
  39. 39. Cell Interactive Glycoproteins• Bind to both cells and ECM• Fibronectin (RGDS, REDV)• Laminin (YIGSR, IKVAV, PDSGR)• Vitronectin (RGDV)
  40. 40. Integrins• Communication channels for cells• Cell cell and cell matrix binding• Bind to cell surface receptors
  41. 41. Growth Factors• Found in vitro that application of certain proteins applied to wounds accelerate normal rate of healing• Important to process of wound healing
  42. 42. • Most important biologically active group of molecules to be identified• Generally small to medium sized proteins and glycoproteins• Mediate potent biological effects on all cell types• Involved in all physiological processes
  43. 43. Cytokines• Interleukins• Interferons• Cytotoxins• Colony Stimulating Factors• Growth Factors• Suppressor, Inhibitory Factors
  44. 44. • Stimulate or inhibit – Cell proliferation – Differentiation – Migration – Adhesion – Gene expression – Secretion and action of other growth factors• Different growth factors share the same biological effects
  45. 45. • Most show more than one property and are able to mediate vast array of biological functions (pleiotropic)• Currently 100+ have been discovered, 20 different families based on structural homology• Not stored as preformed molecules• Require proteolytic activation• May need to bind to ECM for activity and stabilization
  46. 46. • Synthesis is initiated by new gene transcription• Act by binding to cell surface receptors• Important autocrine and paracrine regulators of cell growth and function• Names indicative of original location of discovery, not range of potential effects• Characterized by short biological half lives (PDGF, 2 minutes in blood for example)
  47. 47. Epidermal Growth Factor• Most characterized growth factor• 53 amino acids, 6 kDa• Stimulatory for wide variety of cell types• Initial changes include – Increase in active transport of low MW compounds – Protein phosphorylation – Membrane translocation – Receptor internalization
  48. 48. EGF diagram
  49. 49. The EGF Receptor as a Model
  50. 50. Receptor Ligand Binding• Often monitored using 125I• Incubation of cells with ligand for specified time• Rapid removal of unbound ligand• Measurement of radioactivity• Non specific binding is measured by adding high concentrations of unlabeled growth factor to system
  51. 51. Specific binding diagram
  52. 52. Receptor + Ligand diagram kf R + L↔C krkf R + L ↔C kr kr KD = kf RL C= KD
  53. 53. • KD is equilibrium dissociation constant• Small KD, high KA (KD-1), equilibrium association constant, means high affinity of receptor for ligand• High affinity KD = 10-15• Low affinity KD = 10-6• Function of temperature, pH
  54. 54. Cooperativity• Binding constants – KD and one or both of kr and kf – vary with extent of receptor occupancy
  55. 55. • Believed that EGF and receptor are monovalent• EGF receptor thought to be able to dimerize in some studies• Dimerization seems to be enhanced by presence of EGF• Affinity of EGF for dimerized receptors possibly higher than for monomeric receptors• Mathematical model allows understanding of complex surface interactions
  56. 56. Receptor Ligand Trafficking
  57. 57. Receptor Downregulation• Can lead to receptor downregulation• Essentially loss of cell surface receptors – Endocytotic (internalization step) – Sorting – Synthetic
  58. 58. Cells• Identification of a cell source remains a significant problem• In some cases ingrowth of host cells can lead to the generation of new tissue• In most cases difficult to obtain adequate numbers of cells in order to maintain cellular function• Stem cells are a possibility