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
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
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
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)
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
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)
55.
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
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
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
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
69. Receptor Downregulation
• Can lead to receptor downregulation
• Essentially loss of cell surface
receptors
– Endocytotic (internalization step)
– Sorting
– Synthetic
70.
71.
72.
73.
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