Tissue Engineering

Tissue Engineering is the in vitro development (growth) of tissues

or organs to replace or support the function of defective or injured
body parts.
Research is presently being conducted on
several different types of tissues and organs,
including:
Skin
Cartilage
Blood Vessels
Bone
Muscle
Nerves
Liver
Kidney
etc. etc. etc.

Tissue Organization
Before a tissue can be developed in vitro, first we must understand how
tissues are organized. The basic tenant here is that:

all tissues are comprised of

several levels of structural hierarchy
These structural levels exist from the macroscopic level (centimeter range)
all the way down the molecular level (nanometer range)

there can be as many as 7-10 distinct levels of structural organization in

some tissues or organs

Organization of the Tendon

Organization of the Kidney

Cellular Communications
Soluble Signals:

small proteins (15-20 kDa) which are chemically stable with long half-lives
(unless specifically degraded)

growth factors, steroids, hormones, cytokines, chemokines

bind to membrane receptors usually with high affinity (low binding constants:
10-100 pM)
can diffusion long distances

Cellular Communications
Cell-to-Cell Contact:

some membrane receptors are adhesive molecules

adherent junctions and desmosomes

other serve to create junctions between adjacent

cells allowing for direct cytoplasmic communication
gap junctions
1.5-2 nm diameter and only allow transport of small
molecules ~1 kDa

Cellular Communications
Cell-ECM Interactions:

ECM is multifunctional and also provides a substrate that cells can

communicate
since cells synthesize the ECM, they can modify the ECM to elicit specific
cellular responses
several specialized receptors that allow for cell-ECM interactions
integrins, CD44, etc.
also a mechanism by with cells respond to external stimuli (mechanical
transducers)

Tissue Engineering Scaffolds

Biomaterial Scaffolds Materials:

polymeric
chitosan, alginate, etc.
foams, hydrogels, fibres, thin films

natural
collagen, elastin, fibrin, etc.
hydrogels

ceramic
calcium phosphate based for bone tissue engineering
porous structures

permanent versus resorbable

degradation typically by hydrolysis
must match degradation rate with tissue growth

Chemical and Physical Modifications:

attachment of growth factors, binding sites for integrins, etc.

nanoscale physical features

Tissue Engineering Scaffolds

Culturing of Cells
Types of Cell Culture

monolayer (adherent cells)

suspension (non-adherent cells)
three-dimensional (scaffolds or templates)

Culturing of Cells
Sterilization Methods

ultra-violet light, 70% ethanol, steam autoclave, gamma irradiation, ethylene

oxide gas

Growth Conditions

simulate physiological environment

pH 7.4, 37C, 5% CO2, 95% relative humidity
culture (growth) media replenished periodically

Culture (Growth) Media

appropriate chemical environment

pH, osmolality, ionic strength, buffering agents

appropriate nutritional environment

nutrients, amino acids, vitamins, minerals, growth factors, etc.

Cell Sources
Since the ultimate goal of tissue engineering is to develop replacement
tissue (or organs) for individuals, the use of autologous cells would avoid
any potential immunological complications.
Various classifications of cells used in tissue engineering applications:

primary cells
differentiated cells harvested from the patient (tissue biopsy)
low cellular yield (can only harvest so much)
potential age-related problems

passaged cells
serial expansion of primary cells (can increase population by 100-1000X)
tendency to either lose potency or de-differentiate with too many passages

stem cells

undifferentiated cells
self-renewal capability (unlimited?)
can differentiate into functional cell types
very rare

Stem Cells
Stem cells naturally exist in some tissues (especially those that rapidly
proliferate or remodel) and are present in the circulation.
There are two predominant lineages of stem cells:

mesenchymal
give rise to connective tissues (bone, cartilage, etc.)
although found in some tissues, typically isolated from bone marrow

hematopoietic
give rise to blood cells and lymphocytes
isolated from bone marrow, blood (umbilical cord)

Stem cells are rare; bone marrow typically has:

a single mesenchymal stem cell for every 1,000,000 myeloid cells

a single hematopoietic stem cell for every 100,000 myeloid cells

Stem Cells (Mesenchymal)

Stem Cells (Hematopoietic)

Colony-Forming Units (CFUs)

Bioreactors
a) Spinner Flask:

semi-controlled fluid shear

can produce turbulent eddies which
could be detrimental

b) Rotating Wall

low shear stresses, high mass transfer

rate
can balance forces to stimulate zero
gravity

c) Hollow Fibre

used to enhance mass transfer during

the culture of highly metabolic cells

d) Perfusion

media flows directly through construct

e) Controlled Mechanics

to apply physiological forces during

culture

Bioreactors