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Cellular and Tissue Engineering BIOM9333

Table of Contents

1. INTRODUCTION TO CELLULAR AND TISSUE ENGINEERING ........................................... 4

1.1. Components of tissue engineering system ................................................................................................ 4

1.1.1. Cell source ................................................................................................................................................ 5

1.1.2. Molecules ................................................................................................................................................. 7

1.1.3. Scaffolds ................................................................................................................................................... 8

1.2. Tissue engineering strategies .................................................................................................................... 9

1.2.1. Application of isolated cells ..................................................................................................................... 9

1.2.2. Delivery of cell/tissue inducing agents .................................................................................................. 10

1.2.3. Delivery of cells within matrices (scaffolds) .......................................................................................... 10

1.3. Tissue engineered products ..................................................................................................................... 10

1.4. Obstacles of Tissue engineering .............................................................................................................. 11

1.5. Conclusion ............................................................................................................................................... 11

2. CELL BIOLOGY — STRUCTURE AND FUNCTION ................................................................ 12

2.1. Type of cells ............................................................................................................................................ 12

2.2. Common cell structure ............................................................................................................................ 13

2.2.1. Biomembranes ....................................................................................................................................... 13

2.2.2. Endosome .............................................................................................................................................. 13

2.2.3. Lysosome ............................................................................................................................................... 13

2.2.4. Peroxisome ............................................................................................................................................ 13

2.2.5. Endoplasmic reticulum (ER) ................................................................................................................... 14

2.2.6. Golgi complex ........................................................................................................................................ 14

2.2.7. Nucleus .................................................................................................................................................. 14

2.2.8. Mitochondria ......................................................................................................................................... 14

2.2.9. The cytoskeleton: components and structural functions ...................................................................... 16

2.3. Cell cycle ................................................................................................................................................. 16

2.3.1. Cell division ............................................................................................................................................ 18

2.3.2. Cell death ............................................................................................................................................... 19

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2.4. Molecular motors and the mechanical work of cells ............................................................................... 19

2.4.1. Molecular motors .................................................................................................................................. 19

2.4.2. The mechanical work of cells ................................................................................................................. 20

3. ESSENTIALS BIOCHEMISTRY ................................................................................................... 21

3.1. Chemical building blocks of cells ............................................................................................................. 21

3.1.1. Proteins .................................................................................................................................................. 21

3.1.2. Nucleic acids .......................................................................................................................................... 23

3.1.3. Polysaccharides ...................................................................................................................................... 29

3.2. Membrane Transport .............................................................................................................................. 29

3.2.1. Passive diffusion .................................................................................................................................... 29

3.2.2. Membrane proteins mediate transport ................................................................................................. 30

3.2.3. Osmotic Pressure ................................................................................................................................... 31

4. EXTRACELLULAR MATRIX SCAFFOLDS ................................................................................ 32

4.1. ECM components .................................................................................................................................... 32

4.1.1. Fibrous proteins ..................................................................................................................................... 32

4.1.2. Non-fibrous – Proteoglycans ................................................................................................................. 34

4.2. Integrin-containing adhesive structures .................................................................................................. 37

4.3. Focal adhesion ........................................................................................................................................ 37

4.4. Cell Junctions .......................................................................................................................................... 38

4.4.1. Tight Junctions ....................................................................................................................................... 38

4.4.2. Anchoring junctions ............................................................................................................................... 38

4.4.3. Communicating Junctions ...................................................................................................................... 38

4.5. Cell Locomotion ...................................................................................................................................... 39

5. CELL LINEAGE ............................................................................................................................... 40

5.1. Evolution of a cell line: ............................................................................................................................ 40

5.2. Advantages and disadvantage of tissue Culture ...................................................................................... 41

5.3. Initiation of cell culture ........................................................................................................................... 41

6. POLYMERIC SCAFFOLDS FOR TISSUE ENGINEERING ...................................................... 43

6.1. Biomaterials for tissue engineering ......................................................................................................... 43

6.2. Properties/design criteria for polymers in tissue engineering ................................................................. 43

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6.3. Classification of polymers........................................................................................................................ 44

6.4. Crosslinking ............................................................................................................................................. 45

6.5. Polymer degradation ............................................................................................................................... 45

6.5.1. Degradation types.................................................................................................................................. 45

6.5.2. Results of Degradation........................................................................................................................... 46

6.5.3. Degradation Factors............................................................................................................................... 46

7. CLINICAL APPLICATION ............................................................................................................ 47

7.1. Developing a clinical produce .................................................................................................................. 47

7.1.1. Define the clinical problem .................................................................................................................... 47

7.1.2. Preclinical trial 1: tissue culture testing ................................................................................................. 47

7.1.3. Preclinical trial 2: using animal models .................................................................................................. 47

7.1.4. Clinical trials ........................................................................................................................................... 48

7.2. Tissue regeneration ................................................................................................................................. 49

7.2.1. Factors limiting regeneration................................................................................................................. 49

7.2.2. Regenerative tissue................................................................................................................................ 49

7.3. Product development .............................................................................................................................. 50

7.3.1. Production issues ................................................................................................................................... 50

7.3.2. Examples of current tissue engineering products ................................................................................. 51

7.3.3. Market for tissue engineering products ................................................................................................ 51

7.3.4. Obstacle/challenges of tissue engineering ............................................................................................ 52

7.3.5. Future direction ..................................................................................................................................... 52

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1. Introduction to Cellular and Tissue Engineering

Tissue engineering is the persuasion of the body to heal itself through the delivery to appropriate sites

of molecular signals, cells and supporting structures.

“Tissue engineering is the creation of new tissue for the therapeutic reconstruction of the human body,

by the deliberate and controlled stimulation of selected target cells through a systematic combination of

molecular and mechanical signals” — David F. William

Cellular engineering is the engineering principle and method to understand the biology of cells.

The traditional way of repairing damages on our body is through using medical device(s) that is tailored

to the application based mainly on its mechanical aspects. Problems arise from implantable medical

devices as they are inert, non-responsive to the dynamics ever changing environment of the human

body.

The idea of using tissue engineering (TE) is to persuasion of the body to heal itself using cells, molecules

and scaffolds without implanting any foreign materials. As these implants may wear out before we die

with the increasing mortality rate. The need of TE also due to shortage of donor tissue and organs;

reduction in healing ability caused by conditions such as obesity and osteoporosis; and with no good

alternative therapies are available to repair injuries.

1.1. Components of tissue engineering system

Figure 1.1 Major component of tissue engineering – cells, scaffolds, molecules

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1.1.1. Cell source

Animal

Animal cells are easy sources but there are issues when implanting into humans due to risk of

infections by prion abnormal proteins. In order to use these cells, immunoisolation is usually

required and functional use is not incorporated into the body. E.g. β – cells for pancreas

Human

Differentiated

Autologous cells harvested from foetal and adult tissues/organs such as fibroblasts from skeletal

muscle or spinal cord and chondrocyte. These cells can be used as factories to produce other

types of cells.

Stem Cells

SCs can be derived from the bone marrow, adult tissue, foetal tissue, or embryos.

– Embryonic stem cells

Embryonic stem cells are pluripotent stem cells derived from early embryo, eight cell

stage (blastocyst). Only embryonic SCs are totipotent (potential to develop into ANY cell

type), while the rest are pluripotent (specialized, but have the potential to develop into

a cell type in the same family e.g. blood stem cells).

Advantage: high rate of self renewal can be stimulated to form specific cell types.

Disadvantage: requires presence of feeder layers (usually made of animal cells which

may be transferred during implantation), ethical approval

Improvements: single cells have been able to be extracted from an embryo, allowing for

the embryo's continued survival and growth

– Adults stem cells

Bone marrow

Can be expanded in vitro

Multipotent i.e. differentiate into multiple mesenchymal cell types to form

tissues: cartilage, bone, blood, muscle

May differentiate into non-mesenchymal cell types: neurons, cardiac muscle,

liver

Bioreactors

For isolation, expansion and maintenance

Manipulation in vitro OR enhanced growth in vivo

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Figure 1.2 Cultured cells that have been derived from early human embryos may eventually be coaxed to develop into replacement tissue for a variety of damaged organs.

Figure 1.3 Mesenchymal stem cell

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Induced pluripotent stem cells (iPS)

iPS cells are pluripotent stem cells artificially derived from a non-pluripotent cell. They may allow

researchers to obtain pluripotent stem cells, which are important in research and potentially have

therapeutic uses, without the controversial use of embryos. They may also be less prone to immune

rejection than embryonic stem cells because of the fact that they are derived entirely from the patient.

However, there are still concerns of using retrovirus induced cells to become iPS cells may have the

potential forming teratoma.

Figure 1.4 Cells differentiated from iPS cells

1.1.2. Molecules

Molecules used for TE purposes tend to be growth factors and other such molecules that promote cell

division. Two examples are given as followed:

Vascular Endothelial Growth Factor (VEGF) which can be used to help vascularise heart tissue as an

alternative or in addition to bypass surgery. In the later case, a pellet of VEGF (in this case, VEGF-2) is

implanted into the heart tissue and stimulates the heart vessel to grow towards it.

Platelet Derived Growth Factor (PDGF) which can stimulate the growth of skin tissue, and therefore it's

often upon diabetic patients to fasten the skin repairing process. E.g. PDGF-

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1.1.3. Scaffolds

Human are 3D structures. The repair of the defect requires structure and dimensionality. In order to

achieve that scaffolds are required.

Biological: natural material, cell can interact with the material, can be recognised by cell, non-

toxic, usually poor mechanical properties, comes from biological systems will have batch to

batch variation

Synthetic: controllable and reproducible properties, not recognised by cells, toxicity (include

degraded product)

Design

− Biomimicry: copying nature, biological structures

− Intelligent materials

Surface modification (e.g. growth factors, adhesion molecules)

Bulk modification (e.g. add subunit in polymer can be degraded by biological enzymes)

Control releases (e.g. bioreceptors)

− Fabrication technology

Fused deposition modelling (FDM)

3D printing (bio-plotting)

Nano-spinning

− Biological architecture

Figure 1.5 intelligent materials

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Available scaffold materials

Natural (collagen, coral, chitosan)

Synthetic (PLA, polycaprolactone)

Composite (PLA/alginate)

Permanent

Degradable

Performance

Formed in tissue

1.2. Tissue engineering strategies

1.2.1. Application of isolated cells

Figure 1.6 cell sources for TE

Example: heart failure

Current therapies include medical approach (drugs), stents, grafts

Transplant not typically used but donor shortage is limiting (approx. 2,000 transplants per year

in the USA)

Damaged heart tissue does NOT regenerate

There is a clinical need for new ways of regenerating functioning heart muscle

E.g. Bioheart

− Isolated skeletal muscle myoblasts

− Regeneration of heart muscle

− Treatment for myocardium infraction

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1.2.2. Delivery of cell/tissue inducing agents

Engineer agent i.e. Growth factors (GF)

Deliver agent to target site

E.g. wound healing

skin wounds

platelet derived growth factor (PDGF-ββ)

Regranex

1.2.3. Delivery of cells within matrices (scaffolds)

Open system

Combine cells within scaffolds in a (OPEN) system in which cells are not isolated from host response

Strategy

Engineer scaffold

Combine with isolated cells

Expand in vitro

E.g. Cardiac patch, Orcel

Closed system

Combine cells with matrices in an encapsulated (CLOSED) system in which the immune system cannot

access cells, but necessary functions can continue combine cells with scaffolds in a system

Strategy

Engineer encapsulation/isolation system

Introduce isolated cells (Cell may not have to be autologous)

Systems can be:

− Implanted

− Used as extracorporeal devices

E.g. liver support, pancreas

1.3. Tissue engineered products Skeletal: bone, cartilage, meniscus

Cardiovascular: heart muscle, heart

valves, blood vessels

Skin

Cornea

Dentin

Liver

Pancreas

Kidney

Bladder

Intestine

Nerves

Ureter

Urethra

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1.4. Obstacles of Tissue engineering Vascularisation: most tissues will not grow more than 2mm in diameter without a blood supply

Xenotransplantation: transplantation of cells/tissues/organs from one species to another.

Problems involve rejection, differences in physiology and lifespan, zoonosis (disease

transmission) and various ethical issues.

Gene therapy

Regulatory / low liability

Market need

Financial issue

1.5. Conclusion

Great potential exists for engineered tissues in the treatment of human disease.

As TE products begin to enter the clinic, their future remains unclear.

Justifying cost based on need or superior performance will be key factor in success

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2. Cell Biology — Structure and Function

2.1. Type of cells

Prokaryotes

Before nucleus

Small, relatively simple cells. Most between 1µm and 10 µm

Single cell organisms

May not require oxygen

No organelles (with membranes)

E.g. bacteria, E. coil

Eukaryotes

True nucleus

Larger: 10-100 microns

Often multicellular

Organelles surrounded by membranes

Usually need oxygen

E.g. animals, plants and fungi

Figure 2.1 Prokaryotic cells (a) and eukaryotic cells (b)

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2.2. Common cell structure Take in raw material: nutrient, water, salt

Produce energy

Synthesis molecules

Organise growth

Ability to respond to internal and external changes

2.2.1. Biomembranes

Phospholipids of the composition present in cells spontaneously form sheetlike phospholipid bilayers,

which are two molecules thick. The hydrocarbon chains of the phospholipids in each layer, or leaflet,

form a hydrophobic core that is 3–4 nm thick in most biomembranes. The plasma membrane is made up

of phospholipid bilayer, and each organelle is surrounded by one or more biomembranes.

The lipid bilayer has two important properties,

1. The hydrophobic core is an impermeable barrier that prevents the diffusion of water-soluble

(hydrophilic) solutes across the membrane. Importantly, this simple barrier function is

modulated by the presence of membrane proteins that mediate the transport of specific

molecules across this otherwise impermeable bilayer

2. Stability of the bilayer, the bilayer structure is maintained by hydrophobic and van der Waals

interactions between the lipid chains. Even though the exterior aqueous environment can vary

widely in ionic strength and pH, the bilayer has the strength to retain its characteristic

architecture.

2.2.2. Endosome

Transport protein on the cell plasma membrane mediate the movement of ions and small molecules

across the lipid bilayer, proteins and some other soluble macromolecules in the extracellular milieu are

internalized by endocytosis

2.2.3. Lysosome

Lysosomes are acidic organelles that contain a battery of degradative enzymes, degrading certain

components that have become obsolete for the cell or organism.

Sacs of digestive enzymes budded off the Golgi

Fuse with membrane around debris

2.2.4. Peroxisome

Peroxisomes are a class of roughly spherical organelles, 0.2–1.0μm in diameter. They contain several

oxidases that use molecular oxygen to oxidize organic substances, in the process forming hydrogen

peroxide (H2O2), a corrosive substance. Peroxisomes also contain copious amounts of the enzyme

catalase, which degrades hydrogen peroxide to yield water and oxygen.

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2.2.5. Endoplasmic reticulum (ER)

Smooth endoplasmic reticulum

Synthesis of fatty acids and phospholipids, abundant in hepatocytes to modify or detoxify hydrophobic

chemicals such as pesticides and carcinogens by chemically converting them into more water-soluble,

conjugated products that can be excreted from the body

Packages proteins for transport,

Synthesizes membrane phosolipids

Releases calcium

Detoxification of foreign substances

Rough endoplasmic reticulum

Ribosomes bound to the rough ER synthesize certain membrane and organelle proteins and virtually all

proteins to be secreted from the cell.

Rough due to the presence of ribosomes

Membrane protein synthesis

Extracellular protein synthesis

2.2.6. Golgi complex

Flattened stacks of interconnected membranes

For packaging and distribution of materials to different parts of the cell

2.2.7. Nucleus

The largest organelle in animal cells, is surrounded by two membranes, each one a phospholipid bilayer

containing many different types of proteins. The nucleus is metabolically active, replicating DNA and

synthesizing rRNA, tRNA, and mRNA. Within the nucleus mRNA binds to specific proteins, forming

ribonucleoprotein particles. Most of the cell’s ribosomal RNA is synthesized in the nucleolus. Nuclear

DNA is packaged into chromosomes.

2.2.8. Mitochondria

The main sites of ATP production during aerobic metabolism, it is surrounded by a double membrane,

generate ATP by oxidation of glucose and fatty acids.

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Figure 2.2 ATP is the most common molecule used by cells to capture and transfer energy

ATP production

Figure 2.3 The binding-change mechanism of ATP synthesis from ADP and Pi by the F0F1 complex

Each of the F1 subunits alternate between three conformational states that differ in their binding

affinities for ATP, ADP, and Pi.

Step 1: After ADP and Pi bind to one of the three β subunits (here, arbitrarily designated β1) whose

nucleotide-binding site is in the O (open) conformation, proton flux powers a 120° rotation of the

subunit (relative to the fixed β subunits). This causes an increase in the binding affinity of the β1 subunit

for ADP and Pi to L (low), an increase in the binding affinity of the β3 subunit for ADP and Pi from L to T

(tight), and a decrease in the binding affinity of the β2 subunit for ATP from T to O, causing release of

the bound ATP.

Step 2: The ADP and Pi in the T site (here the β subunit) form ATP, a reaction that does not require an

input of energy, and ADP and Pi bind to the β2 subunit, which is in the O state. This generates an F1

complex identical with that which started the process (left) except that it is rotated 120°.

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Step 3: Another 120° rotation of again causes the O ⟶ L ⟶T ⟶ O conformational changes in the β

subunits described above. Repetition of steps 1 and 2 leads to formation of three ATP molecules for

every 360° rotation of

2.2.9. The cytoskeleton: components and structural functions

The cytosol is a major site of cellular metabolism and contains a large number of different enzymes.

Because of the high concentration of cytosolic proteins, complexes of proteins can form even if the

energy that stabilizes them is weak. The cytosol is highly organized with protein filaments, most soluble

proteins either bound to filaments or otherwise localized in specific regions.

Three types of filaments compose the cytoskeleton

Actin filaments (microfilaments)

− 8–9 nm in diameter

− Have a twisted two-stranded structure

− Supports cell shape, contraction and motility

Microtubules

− Hollow tubelike structures

− 24nm in diameter, whose walls are formed by adjacent protofilaments

− For intracellular transport

Intermediate filaments (IFs)

− 10-nm-diameter rope like structure

− Lend lateral strength to tissue, helps connect cells to tissue

2.3. Cell cycle The simplest type of reproduction entails the division of a parent cell into two daughter cells. A series of

events that prepares a cell to divide followed by the actual division process, called mitosis. Cell cycle

commonly is represented as four stages. The chromosomes and the DNA they carry are copied during

the S (synthesis) phase. The replicated chromosomes separate during the M (mitotic) phase, with each

daughter cell getting a copy of each chromosome during cell division. The M and S phases are separated

by two gap stages, the G1 phase and G2 phase, during which mRNAs and proteins are made. In single-

celled organisms, both daughter cells often (though not always) resemble the parent cell.

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Figure 2.4 During growth, eukaryotic cells continually

progress through the four stages of the cell cycle, generating new daughter cells.

In most proliferating cells, the four phases of

the cell cycle proceed successively, taking from

10–20 hours depending on cell type and

developmental state. During interphase, which

consists of the G1, S, and G2 phases, the cell

roughly doubles its mass. Replication of DNA

during S leaves the cell with four copies of each

type of chromosome. In the mitotic (M) phase,

the chromosomes are evenly partitioned to two

daughter cells, and the cytoplasm divides

roughly in half in most cases. Under certain

conditions such as starvation or when a tissue

has reached its final size, cells will stop cycling

and remain in a waiting state called G0. Most

cells in G0 can reenter the cycle if conditions

change

Figure 2.5 Current model for regulation of the eukaryotic cell cycle

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Passage through the cycle is controlled by G1, S-phase, and mitotic cyclin-dependent kinase complexes

(green). These are composed of a regulatory cyclin subunit and a catalytic cyclin-dependent kinase (CDK)

subunit. Two ubiquitin ligase complexes (orange), SCF and APC, polyubiquitinate specific substrates

including S-phase inhibitors (step 5), securin (step 8), and mitotic cyclins (step 9), marking these

substrates for degradation by proteasomes. Proteolysis of the S-phase inhibitor activates S-phase cyclin-

CDK complexes, leading to chromosome replication. Proteolysis of securing results in degradation of

protein complexes that connect sister chromatids at metaphase, thereby initiating anaphase, the mitotic

period in which sister chromatids are separated and moved to the opposite spindle poles. Reduction in

the activity of mitotic cyclin-CDK complexes caused by proteolysis of mitotic cyclins permits late mitotic

events and cytokinesis to occur. These proteolytic cleavages drive the cycle in one direction because of

the irreversibility of protein degradation. See text for further discussion

2.3.1. Cell division

Figure 2.6 The stages of mitosis and cytokinesis in an animal cell

(a) Interphase (G2): DNA and centrosome replication. After DNA replication during the S phase, the

chromosomes, each containing a sister chromatid, are decondensed and not visible as distinct structures.

By G2 the centrioles have replicated to form daughter centrosomes. (b) Prophase: centrosome migration.

The centrosomes, each with a daughter centriole, begin moving toward opposite poles of the cell. The

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chromosomes begin to condense, appearing as long threads. (c) Prometaphase: spindle formation. The

nuclear envelope fragments into small vesicles and spindle microtubules enter the nuclear region.

Chromosome condensation is completed; each visible chromosome is composed of two chromatids held

together at their centromeres. Kinetochores at centromeres attach chromosomes to spindle

microtubules. (d) Metaphase: chromosome alignment. The chromosomes move toward the equator of

the cell, where they become aligned in the equatorial plane. (e) Anaphase: chromosome separation. The

two sister chromatids separate into independent chromosomes. Each chromosome, attached to a

kinetochore microtubule, moves toward one pole. Simultaneously, the poles move apart. (f) Telophase

and cytokinesis. Nuclear membranes re-form around the daughter nuclei; the chromosomes

decondense and become less distinct. The spindle disappears as the microtubules depolymerize, and cell

cleavage proceeds. (g) Interphase (G1): Following cleavage, the daughter cells enter G1 of interphase

2.3.2. Cell death

Apoptosis is programmed cell death and is essential in controlling multicellular development

(during morphogenesis of human hand, apoptosis mediates separation of developing digits from

a fin-like bud). It is different from necrosis and intimately coupled to cell proliferation.

Necrosis is cell death from injury resulted from external environmental insult, which is a

pathological process. cells that undergo this process swell and burst, releasing their intracellular

contents, which can damage surrounding cells and frequently cause inflammation

The genes involved in controlling cell death encode proteins with three distinct functions:

Killer proteins are required for a cell to begin the apoptotic process.

Destruction proteins do things like digest DNA in a dying cell.

Engulfment proteins are required for phagocytosis of the dying cell by another cell

2.4. Molecular motors and the mechanical work of cells

2.4.1. Molecular motors

Specialized enzymes, motor proteins generate the forces necessary for many cellular movements. These

mechanochemical enzymes convert energy released by the hydrolysis of ATP or from ion gradients into

a mechanical force. Motor proteins generate either linear or rotary motion. Some motor proteins are

components of macromolecular assemblies, but those that move along cytoskeletal fibers are not. This

latter group comprises the myosins, kinesins, and dyneins—linear motor proteins that carry attached

“cargo” with them as they proceed along either microfilaments or microtubules. DNA and RNA

polymerases also are linear motor proteins because they translocate along DNA during replication and

transcription.

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Properties of motor proteins

The ability to transduce a source of energy, either ATP or an ion gradient, into linear or rotary

movement

The ability to bind and translocate along a cytoskeletal filament, nucleic acid strand, or protein

complex

Net movement in a given direction

2.4.2. The mechanical work of cells

Figure 2.7 Operational model for the coupling of ATP hydrolysis to movement of myosin along an actin filament

The cycle for a myosin II head that is part of a

thick filament in muscle, but other myosins that

attach to other cargo (e.g., the membrane of a

vesicle) are thought to operate according to the

same cyclical mechanism. In the absence of

bound nucleotide, a myosin head binds actin

tightly in a rigor state.

Step 1: Binding of ATP opens the cleft in the

myosin head, disrupting the actin-binding site

and weakening the interaction with actin.

Step 2: Freed of actin, the myosin head

hydrolyzes ATP, causing a conformational

change in the head that moves it to a new

position, closer to the (+) end of the actin

filament, where it rebinds to the filament.

Step 3: As phosphate (Pi) dissociates from the

ATP-binding pocket, the myosin head

undergoes a second conformational change—

the power stroke— which restores myosin to its

rigor conformation. Because myosin is bound to

actin, this conformational change exerts a force

that causes myosin to move the actin filament.

Step 4: Release of ADP completes the cycle

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3. Essentials Biochemistry

3.1. Chemical building blocks of cells

3.1.1. Proteins

Proteins are linear polymers containing ten to several thousand amino acids linked by peptide bonds.

The monomeric building blocks of proteins are 20 amino acids, all of which have a characteristic

structure consisting of a central carbon atom (C) bonded to four different chemical groups: an amino

(NH2) group, a carboxyl (COOH) group, a hydrogen (H) atom, and one variable group, called a side chain,

or R group. Because of the carbon in all amino acids except glycine is asymmetric.

Figure 3.1 Common structure of amino acids

The carbon atom (C) of each amino acid is

bonded to four chemical groups. The side chain,

or R group, is unique to each type of amino acid.

Because the C in all amino acids, except glycine,

is asymmetric, these molecules have two mirror

image forms, designated L and D. Although the

chemical properties of such optical isomers are

identical, their biological activities are distinct.

Only L amino acids are found in proteins

Protein structures

Primary protein structure

− Linear or sequence arrangements of the amino acid residues

− Short chain of amino acids (20–30 amino acid residues) linked together to form peptide,

then peptide forms polypeptide (protein) contain as many as 4000 residues

− Average molecular weight of amino acids in proteins is 113

Secondary protein structure

− Various spatial arrangements resulting from the folding of localized parts of a polypeptide

chain

− Stabilized hydrogen bonds form between certain residues, parts of the backbone fold into

one or more well-defined periodic structures: the alpha (α) helix, the beta (β) pleated sheet,

or a short U-shaped turn

Tertiary protein structure

− Structure stabilized by hydrophobic interactions between the nonpolar side chains,

hydrogen bonds between polar side chains, and peptide bonds

− Compactly together in turns and random coils of the α helices and the β pleated sheet

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Quaternary protein structure

− Multimeric proteins consist of two or more polypeptides or subunits

− Very large, exceeding 1mDa in mass

− Approaching 30–300nm in size

Folding of Proteins

A polypeptide chain is synthesized by a complex process called translation in which the assembly of

amino acids in a particular sequence is dictated by messenger RNA (mRNA)

Two general families of chaperones are recognised for promoting protein folding probably:

Molecular chaperones, which bind and stabilize unfolded or partly folded proteins, thereby

preventing these proteins from aggregating and being degraded

Chaperonins, which directly facilitate the folding of proteins

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Figure 3.2 Chaperone- and chaperonin-mediated protein folding

(a) Many proteins fold into their proper three dimensional structures with the assistance of Hsp70-like

proteins (top). These molecular chaperones transiently bind to a nascent polypeptide as it emerges from

a ribosome. Proper folding of other proteins (bottom) depends on chaperonins such as the prokaryotic

GroEL, a hollow, barrel-shaped complex of 14 identical 60,000-MW subunits arranged in two stacked

rings. One end of GroEL is transiently blocked by the cochaperonin GroES, an assembly of 10,000-MW

subunits. (b) In the absence of ATP or presence of ADP, GroEL exists in a “tight” conformational state

that binds partly folded or misfolded proteins. Binding of ATP shifts GroEL to a more open, “relaxed”

state, which releases the folded protein

3.1.2. Nucleic acids

Nucleic acids are linear polymers containing hundreds to millions of nucleotides linked by

phosphodiester bonds. The monomers from which DNA and RNA are built, called nucleotides, all have a

common structure: a phosphate group linked by a phosphoester bond to a pentose (a five-carbon sugar

molecule) that in turn is linked to a nitrogen- and carbon-containing ring structure commonly referred to

as a base. The bases adenine, guanine, and cytosine are found in both DNA and RNA; thymine is found

only in DNA, and uracil is found only in RNA.

A single nucleic acid strand has a backbone composed of repeating pentose-phosphate units from which

the purine and pyrimidine bases extend as side groups. A nucleic acid strand has an end-to-end chemical

orientation: the 5’ end has a hydroxyl or phosphate group on the 5’ carbon of its terminal sugar; the 3’

end usually has a hydroxyl group on the 3’ carbon of its terminal sugar.

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Figure 3.3 Chemical structures of the principal bases in nucleic acids

DNA (deoxyribonucleic acid)

DNA contains all the information required to build the cells and tissues of an organism

DNA is arranged in hereditary units, now known as genes

In the process of transcription, the information stored in DNA is copied into ribonucleic acid

(RNA), which has three distinct roles in protein synthesis.

DNA consists of two associated polynucleotide strands that wind together to form a double helix.

The orientation of the two strands is antiparallel.

The strands are held in precise register by formation of base pairs between the two strands: A is

paired with T through two hydrogen bonds; G is paired with C through three hydrogen bonds.

DNA synthesis always proceeds in the 5’⟶3’ direction because chain growth results from

formation of a phosphoester bond between the 3’ oxygen of a growing strand and the α

phosphate of a deoxynucleoside 5’-triphosphate precursor (dNTP)

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Figure 3.4 The DNA double helix

(a) Space-filling model of B DNA, the most common form of DNA in cells. The bases (light shades) project

inward from the sugar-phosphate backbones (dark red and blue) of each strand, but their edges are

accessible through major and minor grooves. Arrows indicate the 5’⟶3’ direction of each strand.

Hydrogen bonds between the bases are in the center of the structure. The major and minor grooves are

lined by potential hydrogen bond donors and acceptors (highlighted in yellow). (b) Chemical structure of

DNA double helix. This extended schematic shows the two sugar-phosphate backbones and hydrogen

bonding between the Watson-Crick base pairs, A-T and G-C

Figure 3.5 Overview of four basic molecular genetic processes

26

During transcription of a protein-coding gene by RNA polymerase ( ), the four-base DNA code

specifying the amino acid sequence of a protein is copied into a precursor messenger RNA (premRNA) by

the polymerization of ribonucleoside triphosphate monomers (rNTPs). Removal of extraneous

sequences and other modifications to the pre-mRNA ( ), known as RNA processing produces a

functional mRNA, which is transported to the cytoplasm. During translation ( ), the four-base code of

the mRNA is decoded into the 20–amino acid “language” of proteins. Ribosomes, the macromolecular

machines that translate the mRNA code, are composed of two subunits assembled in the nucleolus from

ribosomal RNAs (rRNAs) and multiple proteins (left). After transport to the cytoplasm, ribosomal

subunits associate with an mRNA and carry out protein synthesis with the help of transfer RNAs (tRNAs)

and various translation factors. During DNA replication ( ), which occurs only in cells preparing to

divide, deoxyribonucleoside triphosphate monomers (dNTPs) are polymerized to yield two identical

copies of each chromosomal DNA molecule. Each daughter cell receives one of the identical copies.

RNA (ribonucleic acid)

Messenger RNA (mRNA) carries the genetic information transcribed from DNA in the form of a

series of three nucleotide sequences, called codons, each of which specifies a particular amino

acid

Translation of mRNA is interpreted by transfer RNA (tRNA) with the aid of ribosomal RNA (rRNA),

and its associated proteins. Each type of amino acid has its own subset of tRNAs, which bind the

amino acid and carry it to the growing end of a polypeptide chain if the next codon in the mRNA

calls for it. The correct tRNA with its attached amino acid is selected at each step because each

specific tRNA molecule contains a three-nucleotide sequence, an anticodon, that can base-pair

with its complementary codon in the mRNA.

Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes. These complex

structures, which physically move along an mRNA molecule, catalyse the assembly of amino

acids into polypeptide chains. They also bind tRNAs and various accessory proteins necessary for

protein synthesis. Ribosomes are composed of a large and a small subunit, each of which

contains its own rRNA molecule or molecules

27

28

Figure 3.6 Overview of RNA processing to produce

functional mRNA in eukaryotes

The β-globin gene contains three protein-coding

exons (coding region, red) and two intervening

noncoding introns (blue). The introns interrupt

the protein-coding sequence between the

codons for amino acids 31 and 32 and 105 and

106. Transcription of eukaryotic protein-coding

genes starts before the sequence that encodes

the first amino acid and extends beyond the

sequence encoding the last amino acid,

resulting in noncoding regions (gray) at the ends

of the primary transcript. These untranslated

regions (UTRs) are retained during processing.

The 5’ cap (m7Gppp) is added during formation

of the primary RNA transcript, which extends

beyond the poly(A) site. After cleavage at the

poly(A) site and addition of multiple A residues

to the 3’ end, splicing removes the introns and

joins the exons. The small numbers refer to

positions in the 147–amino acid sequence of β-

globin.

Figure 3.7 The three roles of RNA in protein synthesis

Messenger RNA (mRNA) is translated into

protein by the joint action of transfer RNA

(tRNA) and the ribosome, which is composed of

numerous proteins and two major ribosomal

RNA (rRNA) molecules (not shown). Note the

base pairing between tRNA anticodons and

complementary codons in the mRNA. Formation

of a peptide bond between the amino group N

on the incoming aa-tRNA and the carboxyl-

terminal C on the growing protein chain (purple)

is catalyzed by one of the rRNAs.

29

3.1.3. Polysaccharides

Polysaccharides are linear or branched polymers of monosaccharides (sugars) such as glucose linked by

glycosidic bonds. The building blocks of the polysaccharides are the simple sugars, or monosaccharides.

Monosaccharides are carbohydrates, which are literally covalently bonded combinations of carbon and

water in a one-to-one ratio (CH2O)n.

3.2. Membrane Transport

3.2.1. Passive diffusion

Molecules can diffuse across cellular membranes without the aid of transport proteins. No metabolic

energy is expended because movement is from a high to a low concentration of the molecule, down its

chemical concentration gradient. A simple membrane bilayer is permeable to small hydrophobic

molecules and small uncharged polar molecules, slightly permeable to water and urea, and essentially

impermeable to ions and to large polar molecules

Figure 3.8 Relative permeability of a pure phospholipid bilayer to various molecules

30

3.2.2. Membrane proteins mediate transport

Transport of most molecules into and out of cells requires the assistance of specialized membrane

proteins.

Figure 3.9 Membrane transport proteins

1. Pumps utilize the energy released by ATP hydrolysis to power movement of specific ions (red

circles) or small molecules against their electrochemical gradient

2. Channels permit movement of specific ions (or water) down their electrochemical gradient.

3. Transporters, which fall into three groups, facilitate movement of specific small molecules or

ions.

3a. Uniporters transport a single type of molecule down its concentration gradient.

3b, c. Cotransport proteins (symporters and antiporters) catalyze the movement of one

molecule against its concentration gradient (black circles), driven by movement of one or more

ions down an electrochemical gradient (red circles).

Active transport

ATP-powered pumps (or simply pumps) are ATPases that use the energy of ATP hydrolysis to move ions

or small molecules across a membrane against a chemical concentration gradient or electric potential or

both.

Facilitated diffusion

Channel proteins transport water or specific types of ions and hydrophilic small molecules down their

concentration or electric potential gradients

Co-transporters

Transporters (also called carriers) move a wide variety of ions and molecules across cell membranes.

Three types of transporters have been identified,

Uniporters transport a single type of molecule down its concentration gradient via facilitated

diffusion.

Antiporters and symporters couple the movement of one type of ion or molecule against its

concentration gradient with the movement of one or more different ions down its concentration

gradient

31

Rate of transport

Figure 3.10 Rate of transport versus concentration

difference plot

Lipid diffusion shows a linear

relationship. Rate of diffusion increases

with concentration gradient

Facilitated diffusion: maximum rate –

limited by number of transport proteins

(saturation)

Rate of active transport increases with

concentration gradient

− High rate even when no

concentration difference across the

membrane

− Stops if cellular respiration stops

(cells die)

3.2.3. Osmotic Pressure

The pressure that must be applied to a solution to prevent the inward flow of water across a semi-

permeable membrane

Hypertonicity is the presence of a solution that causes cells to shrink

Hypotonicity is the presence of a solution that causes cells to swell

Isotonic is the presence of a solution that produces no change in cell volume

32

4. Extracellular Matrix Scaffolds

Cells don’t end at their plasma membrane. Cells secrete, assemble and live in a matrix

ECM: meshwork of macromolecules outside plasma membrane

Facilitates communication into/out of cell

− Stores and presents regulatory molecules

− May be connected to the cytoplasm via integrin proteins present in the plasma membrane

− Binds cells together in tissues

Provides structural support and architecture

− Mechanical support – structural integrity, tissue architecture

− Cell differentiation cues

− Controls cell growth and migration

− Determines spatial orientation

Consists mainly of glycoprotein ( proteins with oligosaccharide chains), especially collagen

Some cells attach directly to ECM by bonding to collagen or fibrinogen

4.1. ECM components

4.1.1. Fibrous proteins

Collagen

Major scaffold protein of the ECM and most common forms are type I and III (Type XVIII is a

proteoglycan)

Responsible for functional integrity of tissues such as cartilage, skin, tendon

High tensile strength, equivalent to steel when compared on cross sectional area

General amino acid sequence: X - Gly – Pro – Hypro – Gly – X arranged in a triple helix

Figure 4.1 The collagen triple helix

(a) Side view of the crystal structure of a polypeptide

fragment whose sequence is based on repeating sets

of three amino acids, Gly-XY, characteristic of

collagen α chains. (Center ) Each chain is twisted into

a left-handed helix, and three chains wrap around

each other to form a right-handed triple helix. The

schematic model (right) clearly illustrates the triple

helical nature of the structure. (b) View down the

axis of the triple helix. The proton side chains of the

glycine residues (orange) point into the very narrow

space between the polypeptide chains in the center

of the triple helix. In mutations in collagen in which

other amino acids replace glycine, the proton in

glycine is replaced by larger groups that disrupt the

packing of the chains and destabilize the triple-

helical structure

33

Elastin

Provides elasticity to tissues

− Due to crosslinking of lysine residues

Produced by fibroblasts and smooth muscle cells

Important for skin, arteries, bladder

Laminin

Located in the basement membrane

Self associates – forms sheets

18 different forms – tissue specific

Binds to cells

34

Fibronectin

300 – 400μg/ml plasma

− Produced by many cells

− Homodimer – 220kD units

Multi domain

− Binds to cells and ECM

Figure 4.2 Organization of fibronectin chains

Vitronectin

Other major cell adhesion protein in blood plasma

− 200 – 400μg/ml in plasma

− Conformationally labile

Also has domain structure

− Binds to cells and ECM

4.1.2. Non-fibrous – Proteoglycans

Proteoglycans proteins with pendant sugar chains

4 families based on sugar structure (glycosaminoglycan)

− Chondroitin sulphate (glucosamine)

− Heparan sulfate (heparin)

− Keratan sulfate

Glycosylation occurs in the golgi

Sugars impart additional biological function – growth factor reservoirs

May contain up to 200 glycosaminoglycan chains

Glycosaminoglycans covalently bound to a core protein via serine side chains

35

Figure 4.3 The repeating disaccharides of glycosaminoglycans (GAGs), the polysaccharide components of proteoglycans

Hyaluronan

viscous, gel–like, compression-resistant

Found in the endothelial cell basement membrane, developing tissue, healing wounds, synovial

fluid

Made by fibroblasts

Degraded in vivo by hyaluronidase

36

Functions:

Lubricant

Shock absorber

Flexible cement

Attachment site

Path for cell migration

Crosslinks to other proteins for matrix stabilisation – heavy chain proteins

Heparin

Molecular weight approximately 1104

20 disaccharide units

Produced and released by mast cells in lungs and liver

Anticoagulant

− Forms a complex with antithrombin III which binds to thrombin and inactivates it

− This leads to limited clot formation

Aggrecan

Approximately 100 GAG chains/molecule, approximately 50 disaccharides/GAG chain

chondroitin sulfate or keratan sulfate

Major GAG–PG in cartilage

− Disperses shocks the joint

− Marker of joint degradation

Syndecans

Family of cell-surface PGs

Core protein domains

− Intracellular

− Transmembrane

− Extracellular

Functions:

Interactions

− Cell-cell

− Cell-matrix

Growth factor receptor

37

4.2. Integrin-containing adhesive structures Many nonepithelial cells have integrin-containing aggregates (e.g., focal adhesions, 3D

adhesions, podosomes) that physically and functionally connect cells to the extracellular matrix

and facilitate inside-out and outside-in signaling.

Integrins exist in two conformations that differ in the affinity for ligands and interactions with

cytosolic adapter proteins

4.3. Focal adhesion Cell adhesion mediated by a localized aggregation of receptors and linking proteins (referred to

as focal adhesion complexes or FACs)

FAC formation is dynamic, not static

KD for integrin-ECM protein binding approximately 10-6 to 10-7M

− Low affinity binding ⇒ easily reversible

− Facilitate cell migration by FAC formation/deformation

Cell signalling

Between cells and the body to coordinate the number of cells, their position and function

Cells communicate through chemical signals which direct cell proliferation, differentiation,

migration, cytokine production or ECM production

As the gene expression provided of every cell type is different, cells will response differently to

certain signals.

Complex as cells may be exposed to many signals at any one time

Signals may be

− Diffusible molecules

− Extracellular matrix proteins

− Membrane-bound ligands

Cells communicate by direct contact with

proteins expressed on the membrane of both

cells. One protein acts as a ligand and the other

as a receptor. Binding of the proteins causes a

change in conformation leading to activation

which transduces the signal into the cell e.g.

receptor tyrosine kinases, G proteins, integrins

38

4.4. Cell Junctions

4.4.1. Tight Junctions

Seals neighbouring cells together in an epithelial sheet

4.4.2. Anchoring junctions

Adheren junctions: Joins actin bundles between neighbouring cells

Focal adhesion: Attaches actin filaments in a cell to the ECM

Desmosome: Joins intermediate filaments between neighbouring cells

Hemidesmosome: Anchors intermediate filaments in a cell to the ECM

4.4.3. Communicating Junctions

Gap junctions: Cell-cell junction allows the passage of small water-soluble ions, molecules and

electrical signals.

Chemical synapse: Neurotransmission

Figure 4.4 The principal types of cell junctions that connect the columnar epithelial cells lining the small intestine

(a) Schematic cutaway drawing of intestinal epithelial cells. The basal surface of the cells rests on a basal

lamina, and the apical surface is packed with fingerlike microvilli that project into the intestinal lumen.

Tight junctions, lying just under the microvilli, prevent the diffusion of many substances between the

intestinal lumen and the blood through the extracellular space

39

4.5. Cell Locomotion

Cell locomotion results from the coordination of motions generated by different parts of a cell. These

motions are complex.

Membrane Extension: The network of actin filaments at the leading edge is a type of a cellular engine

that pushes the membrane forward by an actin polymerization–based mechanism. The concerted action

of numerous filaments undergoing similar movements and their cross-linkage into a mechanically strong

network generate sufficient force (several piconewtons) to push the membrane forward.

Cell–Substrate Adhesions: When the membrane has been extended and the cytoskeleton has been

assembled, the membrane becomes firmly attached to the substratum. Time-lapse microscopy shows

that actin bundles in the leading edge become anchored to the attachment site, which quickly develops

into a focal adhesion. The attachment serves two purposes: it prevents the leading lamella from

retracting and it attaches the cell to the substratum, allowing the cell to push forward.

Cell Body Translocation: After the forward attachments have been made, the bulk contents of the cell

body are translocated forward.

Breaking Cell Attachments: Finally, in the last step of movement (de-adhesion), the focal adhesions at

the rear of the cell are broken and the freed tail is brought forward.

Figure 4.5 Steps in keratinocyte movement

In a fast moving cell such as a fish epidermal cell, movement begins with the extension of one or more

lamellipodia from the leading edge of the cell (1); some lamellipodia adhere to the substratum by focal

adhesions (2). Then the bulk of the cytoplasm in the cell body flows forward (3). The trailing edge of the

cell remains attached to the substratum until the tail eventually detaches and retracts into the cell body

(4).

40

5. Cell Lineage Cell culture: cells (usually derived from multicellular eukaryote)are grown under controlled

conditions

Primary culture: growth of cells from explanted tissue

Passage: dispersing and moving cells to fresh vessels

“Contact inhibited” once cells cover entire surface because cells don’t need to grow anymore

Cell line: formed after the first passage

Monolayer culture: growth of cells on a solid substrate (common)

Suspension culture: growth of cells without support

Medium: liquid which supports cell growth- often contains calf serum

Medium provides control of pH, buffering components, glucose, etc

Issue with calf serum is that we don’t really know what’s in calf serum or how it changes from

batch to batch.

Confluence: growth area covered with a layer of touching cells

5.1. Evolution of a cell line: Within the first two weeks cell number drops due to the shock of the cells being removed from

in vivo environment

Exponential growth of the cell line until a plateau

Brought about through apoptosis, cells have a certain life cycle

In order to go past the plateau - transformation occurs:

− Tumours and cancerous cells - continuous cell line

− Vectors used to induce this type of cell line are viruses

Cell death

Figure 5.1 Evolution of a Cell Line

41

5.2. Advantages and disadvantage of tissue Culture

Advantages of tissue Culture

Control of Environment Temperature, pH, hormone and nutrient concentrations.

Control of Cellular Constitution Defined cellular identity and controlled cellular interactions.

Less Time Time measured in days to weeks, rather than months.

Scale: Less Reagent Required Distribution of compounds in milliliter volumes compared to systemic distribution.

Less Expensive Than In Vivo

Experiments

After initial equipment acquisition, the cost of consumables and media are

significantly less expensive than animal husbandry.

Replicates and Variability Traditionally easy to replicate and less variability than in vivo studies.

Reduction of Animal Use Treatment is administered to cell lines or primary cultures/tissue rather than in the

intact animal. Animals stress due to treatment is reduced.

Stocking Suspend cells in DMSO and cryogenically stocked down in liquid nitrogen for later

use

Disadvantages of tissue Culture

Lack of Systemic Input from the

Periphery

Brain pathology receives input from the peripheral system.

Sterile Technique/Expertise The process of cell culture requires knowledge about sterile technique and the cell

types of interest.

Potential for Dedifferentiation and

Selection

The cell types may not be identical to cells in the intact system.

Three Dimensional Structure Lost The effects of tissue and organ structure are not present.

5.3. Initiation of cell culture There are 3 common ways to start a tissue culture:

Organ culture: 3D architectural characteristics of the tissue are retained and cultured either on a

grid, in gel or on a raft at the gas-liquid interface. Cell proliferation is limited.

Primary explants culture: a piece of tissue is cultured on a solid substrate at either glass- or

plastic - liquid interface.

Cell culture: mechanically or enzymatically release the cells in suspension which can be cultured

as an adherent monolayer on a solid substrate or a suspension in culture medium. Good

proliferation is usually found for such tissue culture. Propagation of cells becomes feasible and

requires passage and culturing of the daughter cells. This forms the beginning of a cell line.

42

Figure 5.2 Types of tissue culture

43

6. Polymeric Scaffolds for Tissue Engineering

Biocompatibility: appreciate or suitable biological performance of a material in a specific

application

“The ability of a material to perform with an appropriate host response in a specific application”

— The Williams Definition of Biocompatibility

Biological performance: result of the interaction between biomaterials and living system;

encompasses both the host response and the material response

Host response: the local and systemic response, other than the intended therapeutic response

of living system to the material

Material response: the response of the biomaterial to the living system or how the biomaterial is

altered following contact with the living system

Polymers are macromolecules built up by linking large numbers of much smaller molecules

− Small molecules = Monomers

− Reactions of small molecules = Polymerization

Polymer scaffolds can be used in tissue engineering applications to:

Deliver cells and/or signals

Define or fill space for engineered tissues

Guide tissue development

Completely degrade in vivo

6.1. Biomaterials for tissue engineering Polymers

− Natural

− Synthetic

Metals

Ceramics

Composites

6.2. Properties/design criteria for polymers in tissue engineering Strength

Stiffness

Thermal

Surface

Electrical

Economics

Scale-up

Environmental

Aesthetic

Safety

44

When it comes to selecting an appropriate polymer for use as a TE scaffold (or any material for that

matter) it is important to realise that there are many properties that must be considered.

These include, but are not limited to the following:

Appropriate physical/mechanical properties

− Strength, elasticity, porosity, etc

Production properties

− Can it be purified, fabricated and sterilized easily

Does not induce undesirable host reactions

− Toxicity, thrombus, etc

Maintain properties during sterilization and use

− Indirect to direct contact

− Over time period form 1 hr, 1 day, 1 year, 10 years

"Ideal" OPEN scaffold design criteria

Biocompatible

Completely resorbable

Non-toxic degradation products

Support differentiated cell proliferation and function

Adequate shelf life

"Ideal" CLOSED scaffold design criteria

Biocompatible

Non-degradable

Non-toxic leachable products

Support differentiated cell proliferation and function

Prevent cell ingrowth/act as an immune-isolation barrier

Adequate shelf life

6.3. Classification of polymers

45

6.4. Crosslinking

Physical

Hydrogen Bonding

Freeze Thawing

− Inducing crystallinity

Entanglements

Chemical

Condensation

Free Radical

6.5. Polymer degradation

6.5.1. Degradation types

Chemical/Molecular

Hydrolysis

H2O attacks certain bonds in the chains,

causes chain cleavage

Oxidation

Enzymes/peroxides attack susceptible

bonds, causes chain cleavage

Macroscopic Bulk

Fatigue

Repeated movement of device causes

breakdown of device

Wear

Constant friction causes pieces of device to

break off

Stress

Constant stress can cause deep surface

cracks in the device

46

6.5.2. Results of Degradation

Decrease in molecular weight

Mechanical properties decline

Swelling

Network Deterioration

Weight Loss

Branching or crosslinking

Surface cracking

Embrittlement of polymer

6.5.3. Degradation Factors

Choice of polymer

− Chemistry, morphology

Processing

− Fabrication, geometry

Additives

− Stabilizers or leachables

Cleaning, packaging, sterilization and

transport

Moisture

− Possible leaching, swelling, softening or

hydrolysis

Proteins

− Attachment to the surface, fouling,

degradation

Temperature

− Thermal transitions, changes in

mechanics, degradation

pH

− Changes in mechanics, swelling,

degradation

Oxidation

Radiation

UV, gamma rays

Overuse / Wear / Fatigue

Stresses

Tensile, compressive, shear

Misuse

47

7. Clinical Application

7.1. Developing a clinical produce

7.1.1. Define the clinical problem

Understand what the current available technology is and where it might head in the future. Looking at

what is product available now and what can be available in the future. Also understand what the market

need is.

Knowledge about the tissue/organ

Gross anatomy (macroscopic anatomy): size, shape, location, structure

Histology (microscopic anatomy): morphology, microscopic structure, cellular level

Physiology: function (macroscopic, microscopic)

Pathology: abnormalities or diseases (long term or short term impact on the body)

Current clinical treatments (evaluating the need for tissue engineered products compare to the

current treatment method based on availability, complexity, functions, cost, ethics, side effect,

etc)

Key issues

Types of tissue/organ going to be engineered (major group of tissue e.g. connective tissue,

epithelium tissue, muscle tissue)

Location and specific features that distinguish it from other members of the same tissue

category

Function of where the tissue is located and degree of lost in function

The level of regeneration/regrowth that requires to regain meaningful function

7.1.2. Preclinical trial 1: tissue culture testing

Why use tissue culture?

Easy to control variables and its environment

Can run multiple samples

Fast results

To reduce the number of animal used (ethical reason, financial problems, complications rise

from using animal models, etc)

7.1.3. Preclinical trial 2: using animal models

Why use animal models?

Modeling in 3 dimensions

Dynamic system that has mechanical forces acting on it, complete endocrine/exocrine system

(e.g. hormone regulation), has paracrine signaling (cell signaling), complete nerve systems

48

Difference in vitro study and in vivo study (animal models)

Tissue culture Animal models

Use cell culture medium Vascularised Use cell culture medium Have lymphatic system

Not applicable Have cell signaling (paracrine) system Use cell culture medium Have blood Use cell culture medium Have WBC and RBC

Uses buffers Have regulation system (pH, electrochemical) Not applicable Have nervous system Not applicable Enable fibrous thrombus formation

Not grown under stress loading Complex mechanical loading for every

tissue/organ

7.1.4. Clinical trials

Applying the understanding from the animal models into the clinical situation

Understand the differences between human and animals

Difference between humans and animals

Difference in gross anatomy

Difference in tissue histology, need to know the human equivalent of the animal tissue

Difference in motion and movement, need to know the applied loading and function of the

animal to human equivalent

Sex differences of animal, different hormones in different sex may cause big effect responses in

animals

Animals have different time frame of age compare to human

Diseases in animals may have a different anomalies which may make it hard to interpret the

result correctly (e.g. genetic anomalies)

Different evaluation of pain responses compare to human

Studies can be done on animal models

Response of the normal or induced disease tissue to the implantation, conclusions can be drawn

from the control to the test samples (e.g. integration test by histology)

Functional effects (e.g. mechanical loadings, blood sample tests) on the implant

(Feedback from pain response is very hard as there are no indicators for pain response in animals)

Feedback from animal models

Safety issue

Local and systemic response to the construct (host response)

Material response caused host response

Toxicity responses (in vitro: cytotoxicity; in vivo: systemic toxicity)

49

Effectiveness of the treatment

Assessing the function of the tissue/organ regenerated (e.g. bone repair: testing the

strength of the bone, mineral density, cell distribution)

Wound healing time frame

Same as human

Accelerated response (faster than human, scale to human life span)

Regeneration assessment

Histopathological evaluation (e.g. section slide, H&E stain, etc)

Biochemical assessment (e.g. glucose level, insulin level, etc)

Functional properties (e.g. mechanical loading, regrowth, nervous transmission, etc) over

time

Assessing clinical trial data

Pain assessment (use an analogue scale to show the level of pain experienced)

Behavior assessment (adaption of the animal to the implant)

Psychological assessment

Function

Imaging

Non-destructive testing (e.g. indentation probes for mechanical testing)

Biopsies

7.2. Tissue regeneration

7.2.1. Factors limiting regeneration

Maintain cell viability and cell proliferation for thick tissue (problem with transporting nutrient

in and waste material out of the cells in the center of the tissue)

Laboratory engineered tissue lacks vascularisation (problem of transporting nutrient in and

waste product out when implanted in the body if developing vascular system for the implanted

tissue was not fast and sufficient enough)

Collapse of the surrounding tissue into the defect causing the surrounding tissue to be

disorganized and hard to repair structurally

Mechanical stress and strain on the engineered tissue that disestablished the structure during

cell repairing

7.2.2. Regenerative tissue

Tissue can substantially regenerate under certain given signals (e.g. hormones, neurotransmitters,

cytokines, growth factors, etc)

50

Tissue/ organ can regenerate

Bone

Epithelium tissue

Smooth muscle

Tissue/ organ have limited or no regenerate properties

Articular cartilage, ligament, articular disk (e.g. intervertebral disc)

Reason: non vascular, signals from plasma cannot reach the cell to stimulate cell division, low

cell density, low mitotic activity

Large area epithelium tissue damage

Reason: problem with cell migration, infection, exposure to external environment may lead to

dehydration which affects cell survival.

Cardiac, skeletal muscle

Reason: no mitotic activity

Nerve tissue

Reason: no mitotic activity, if nerves grow back function is limited and requires scaffold to

promote cells to connect with each other

7.3. Product development

7.3.1. Production issues

Cell sourcing (mammalian cells: human/animal, commercially bought, from patient)

Controlling cell function (control cell proliferation, retain cellular function:

chondrocyte/fibroblast without differentiating)

Developing interactive biomaterials (bioactive/resorbable material: hydroxyapatite, BioGlass®,

polylactic acid)

Engineering 3D constructs/healing response (tissue vascularisation, resembles the original tissue,

open system scaffold, close system scaffold, maintain cell viability)

Scaling up manufactured products (lab scale to manufacturing scale, batch to batch variation,

automation of the process, process control)

Preserving manufactured products (packaging, shelf life, preserving viable cells)

Controlling in vivo biological responses (response to degradation of product, inflammatory

response)

Engineering immune acceptance

Assessing post-implant viability (evaluating success rate of the implant, biopsies, biochemical

evaluations)

51

7.3.2. Examples of current tissue engineering products

Artificial skin (e.g. Dermagraft®)

Absence of hair follicles, sweat glands, and indentation, it doesn’t require much vascularisation. Product

is less complex, doesn’t have thick 3D tissue problems and much straight forward to produce. Products

provide wound covering and have cells and stimulation for cell proliferation.

Cartilage replacement (e.g. Carticel®)

Non vascular, doesn’t have same structure to normal articular cartilage structure. Used to repair

articular cartilage injuries in adults who have not responded to an arthroscopic or other surgical repair

procedure Autologous cartilage implantation (ACI) method, inject the cartilage into the defect and cover

with periosteal patch to keep chondrocytes in place or fibrous clot. The performance of the implant was

analyzed after a period of time for integration with the surrounding tissue.

Key issues

Cell source and source of type II collagen production

Scaffolds

Integration of the cells with the scaffold and the bone

Mimic mechanical properties of the bone

Cardiovascular

Small diameter blood vessels to carry out blood transport to the cardiac muscle

Heart valves control blood flow

Myocardial patches to repair damaged areas

Key issues

Myocardial repair (cell extraction and cell implantation in clinical trials)

Myocardial patches can only last for only 5 years

Small diameter blood vessel substitute (approx 6mm, none on market)

Current treatment for cardiac blood vessel by,

a) Replace blood vessel from the leg

b) Run out of blood vessel if it needed to be replaced again

c) Overall blood vessels in the body isn’t in good condition

Thrombus formation in small diameter blood vessels

Mechanical issues (continuous pulsation)

Regulatory approval for heart valve (degradation, fatigue, mechanical failure)

7.3.3. Market for tissue engineering products

Current clinical trials on tissue engineered artificial skin, cartilage, bone and bladder repair system

In 2004 the market for TE was USD$144.6m; it is predicted to be USD$2.1b in 2015, which covers skin,

bone, cartilage, dental, cardiovascular, and organ regeneration.

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7.3.4. Obstacle/challenges of tissue engineering

Few commercialized products

High failure rate

High investment, low return

Long term expensive product

Cumbersome regulatory and maintenance requirement

Complex surgical/medical procedure required

Emerging technology, not mature

7.3.5. Future direction

Market going to increase as population ages

Long term focus

Improvement of regulation system to suit TE

Improvement of technology

Application specific material

Artificial scaffold have cues similar to biological systems

Advances in cell technology (e.g. stem cell technology, therapeutic cloning)

Understanding principles

Collaboration with medical society