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1. Tissue Engineering Overview

2. • Can I live with a beating heart that
came from no one?

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. 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. Regulators of
Matrix
Assembly Insoluble
Soluble Matrix Matrix
Molecules Assemblies
CELLS
Matrix Bound
Soluble Growth
Growth
Factors
Factors
Bioactive
Cells
Matrix

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. 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. 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. • 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. • 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. • 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. • 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. 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. The Collagen Molecule
Diagram from Nimni
Diagram page 49 TE book

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. Fibrillogenesis
Figure 9 Nimni

17. Types of Collagen
Figure 11 Nimni

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. Type II Collagen
• Present in large amounts in cartilage
• Also present in intervertebral disk,
vitreous humour of the eye

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. Figure 12 from Nimni

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. 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. Elastin
• Source of elasticity in tissues
• Prominent in lung, skin and blood
wall

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. • 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. 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. • 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. 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. • 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. 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. • 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. • 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. The Glycosaminoglycans
GAG MW A B Sulfates Protein Other Tissues
Sugars
HA 4000 – Glucuronic Glucos- 0 - 0 Skin,
8x106 acid amine vitreous,
cartilage
CS 5000- Glucuronic Galacto 0.2 – 2.3 + Galactos Cartilage
50000 acid s-amine exylose Cornea
Bone
HS 5000- Glucuronic Glucos- 0.2-2.0 + Galactos Lung,
12000 acid amine exylose arteries
KS 4000- Galactose Glucos- 0.9-1.8 + Galactos- Cartilage
19000 amine amine cornea

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. 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. • Potential for limitless heterogeneity
• Can differ markedly in protein
content, molecular size, number and
type of GAGs
• Very difficult to characterize and
classify

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. Cell Interactive Glycoproteins
• Bind to both cells and ECM
• Fibronectin (RGDS, REDV)
• Laminin (YIGSR, IKVAV, PDSGR)
• Vitronectin (RGDV)

40. Integrins
• Communication channels for cells
• Cell cell and cell matrix binding
• Bind to cell surface receptors

49. Growth Factors
• Found in vitro that application of
certain proteins applied to wounds
accelerate normal rate of healing
• Important to process of wound
healing

50. • 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

51. Cytokines
• Interleukins
• Interferons
• Cytotoxins
• Colony Stimulating Factors
• Growth Factors
• Suppressor, Inhibitory Factors

52. • 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

53. • 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

54. • 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)

56. 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

57. EGF diagram

58. The EGF Receptor as a Model

61. 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

62. Specific binding diagram

63. Receptor + Ligand diagram
kf
R + L↔C
krkf
R + L ↔C
kr
kr
KD =
kf
RL
C=
KD

64. • 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

65. Cooperativity
• Binding constants – KD and one or
both of kr and kf – vary with extent of
receptor occupancy

67. • 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

68. Receptor Ligand Trafficking

69. Receptor Downregulation
• Can lead to receptor downregulation
• Essentially loss of cell surface
receptors
– Endocytotic (internalization step)
– Sorting
– Synthetic

74. 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