Tissue Engineering OverviewTissue Engineering OverviewTissue Engineering OverviewTissue Engineering Overview

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

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

• Interdisciplinary field that applies the principle of engineering and life sciences to the development of biological substitutes that restore, maintain or augment tissue function

• Interdisciplinary field that applies the principle of engineering and life sciences to the development of biological substitutes that restore, maintain or augment tissue function

Tissue EngineeringTissue EngineeringTissue EngineeringTissue 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

• 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

InsolubleMatrix

Assemblies

CELLS

Cells

Soluble MatrixMolecules

Regulators ofMatrix

Assembly

Matrix BoundGrowth Factors

BioactiveMatrix

Soluble GrowthFactors

Steps in Tissue EngineeringSteps in Tissue EngineeringSteps in Tissue EngineeringSteps 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

• 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

Extracellular MatrixExtracellular MatrixExtracellular MatrixExtracellular 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

• 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

ECMECMECMECM• Composed of:– Fibrous proteins

• Collagens• Elastin• Fibrillin• Fibronectin• Laminin

– Hydrophilic proteoglycans

• Assembled by cells, modified by cells as they proliferate, differentiate, and migrate

• Composed of:– Fibrous proteins

• Collagens• Elastin• Fibrillin• Fibronectin• Laminin

– Hydrophilic proteoglycans

• Assembled by cells, modified by cells as they proliferate, differentiate, and migrate

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

CollagensCollagensCollagensCollagens

• 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

• 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

Diagram from Nimni

Diagram page 49 TE book

The Collagen MoleculeThe Collagen MoleculeThe Collagen MoleculeThe Collagen Molecule

The Collagen MoleculeThe Collagen MoleculeThe Collagen MoleculeThe Collagen Molecule 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

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

FibrillogenesisFibrillogenesisFibrillogenesisFibrillogenesis

Figure 9 Nimni

Types of CollagenTypes of CollagenTypes of CollagenTypes of Collagen

Figure 11 Nimni

Type I CollagenType I CollagenType I CollagenType I Collagen

• Three chains, two 1 chains, 1 2 chain

• Abundant in skin, tendon, ligament, bone, cornea – 88-99% of total collagen

• Three chains, two 1 chains, 1 2 chain

• Abundant in skin, tendon, ligament, bone, cornea – 88-99% of total collagen

Type II CollagenType II CollagenType II CollagenType II Collagen

• Present in large amounts in cartilage

• Also present in intervertebral disk, vitreous humour of the eye

• Present in large amounts in cartilage

• Also present in intervertebral disk, vitreous humour of the eye

Type III CollagenType III CollagenType III CollagenType 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

• 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

Figure 12 from Nimni

• 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

• 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

Basement Membrane Basement Membrane CollagensCollagens

Basement Membrane Basement Membrane CollagensCollagens

• 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

• 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

ElastinElastinElastinElastin

• Source of elasticity in tissues

• Prominent in lung, skin and blood wall

• Source of elasticity in tissues

• Prominent in lung, skin and blood wall

ElastinElastinElastinElastin• 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

• 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

• 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

• 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

MicrofibrilsMicrofibrilsMicrofibrilsMicrofibrils• 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

• 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

• 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

• 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

Tissue Distribution of Tissue Distribution of Elastic FibresElastic Fibres

Tissue Distribution of Tissue Distribution of Elastic FibresElastic 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

• 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

• 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

• 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

GlycosaminoglycansGlycosaminoglycansGlycosaminoglycansGlycosaminoglycans

• 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

• 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

• 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

• 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

• 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

• 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

The GlycosaminoglycansThe GlycosaminoglycansThe GlycosaminoglycansThe GlycosaminoglycansGAG MW A B Sulfates Protein Other

Sugars

Tissues

HA 4000 – 8x106

Glucuronic acid

Glucos-amine

0 - 0 Skin, vitreous,cartilage

CS 5000-50000

Glucuronic acid

Galactos-amine

0.2 – 2.3 + Galactosexylose

Cartilage

Cornea

Bone

HS 5000-12000

Glucuronic acid

Glucos-amine

0.2-2.0 + Galactosexylose

Lung, arteries

KS 4000-19000

Galactose Glucos-amine

0.9-1.8 + Galactos-amine

Cartilage cornea

ProteoglycansProteoglycansProteoglycansProteoglycans• 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

• 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

ProteoglycansProteoglycansProteoglycansProteoglycans• 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

• 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

• Potential for limitless heterogeneity

• Can differ markedly in protein content, molecular size, number and type of GAGs

• Very difficult to characterize and classify

• Potential for limitless heterogeneity

• Can differ markedly in protein content, molecular size, number and type of GAGs

• Very difficult to characterize and classify

Function of ProteoglycansFunction of ProteoglycansFunction of ProteoglycansFunction 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

• 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

Cell Interactive GlycoproteinsCell Interactive GlycoproteinsCell Interactive GlycoproteinsCell Interactive Glycoproteins

• Bind to both cells and ECM

• Fibronectin (RGDS, REDV)

• Laminin (YIGSR, IKVAV, PDSGR)

• Vitronectin (RGDV)

• Bind to both cells and ECM

• Fibronectin (RGDS, REDV)

• Laminin (YIGSR, IKVAV, PDSGR)

• Vitronectin (RGDV)

IntegrinsIntegrinsIntegrinsIntegrins

• Communication channels for cells

• Cell cell and cell matrix binding

• Bind to cell surface receptors

• Communication channels for cells

• Cell cell and cell matrix binding

• Bind to cell surface receptors

Growth FactorsGrowth FactorsGrowth FactorsGrowth Factors

• Found in vitro that application of certain proteins applied to wounds accelerate normal rate of healing

• Important to process of wound healing

• Found in vitro that application of certain proteins applied to wounds accelerate normal rate of healing

• Important to process of wound healing

• 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

• 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

CytokinesCytokinesCytokinesCytokines

• Interleukins• Interferons• Cytotoxins• Colony Stimulating Factors• Growth Factors• Suppressor, Inhibitory Factors

• Interleukins• Interferons• Cytotoxins• Colony Stimulating Factors• Growth Factors• Suppressor, Inhibitory Factors

• 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

• 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

• 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

• 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

• 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)

• 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)

Epidermal Growth FactorEpidermal Growth FactorEpidermal Growth FactorEpidermal 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

• 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

EGF diagram

The EGF Receptor as a ModelThe EGF Receptor as a ModelThe EGF Receptor as a ModelThe EGF Receptor as a Model

Receptor Ligand BindingReceptor Ligand BindingReceptor Ligand BindingReceptor 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

• 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

Specific binding diagram

Receptor + Ligand diagram

CLRf

r

k

k

D

f

rD

k

k

K

RLC

k

kK

CLRf

r

• 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

• 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

CooperativityCooperativityCooperativityCooperativity• Binding constants – KD and one or

both of kr and kf – vary with extent of receptor occupancy

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

• 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

• 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

Receptor Ligand TraffickingReceptor Ligand TraffickingReceptor Ligand TraffickingReceptor Ligand Trafficking

Receptor DownregulationReceptor DownregulationReceptor DownregulationReceptor Downregulation

• Can lead to receptor downregulation

• Essentially loss of cell surface receptors– Endocytotic (internalization step)– Sorting– Synthetic

• Can lead to receptor downregulation

• Essentially loss of cell surface receptors– Endocytotic (internalization step)– Sorting– Synthetic

CellsCellsCellsCells

• 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

• 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