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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, 37°C, 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