Tissue Engineering

Question 1

Explain what tissue engineering is?

Answer 1

Tissue Engineering is the in vitro development (growth) of tissues or organs to replace or support the function of defective or injured body parts

Question 2

Name the three ways cells can communicate?

Answer 2

1. Soluble signals
2. Cell to Cell contact
3. Cell - ECM interactions

Question 3

What are soluble signals? How does this process work

Answer 3

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)

Diffusion driven process

Question 4

How does cell - to - cell contact work?

Answer 4

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

Question 5

How does ECM interactions work?

Answer 5

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”)

Question 6

What is the role of a scaffold?

Answer 6

substrate material must be inserted to aid in organization of the cells in three dimensions

Question 7

What are the three types of scaffold material? and give examples

Answer 7

1. Polymeric e.g. Chitosan, Alginate, Foams, Hydrogels
2. Natural e.g.Collagen, Elastin, Fibrin a.k.a Factor Ia
3. Ceramic e.g. calcium phosphate based for bone tissue engineering
   Calcium phosphate ceramics (CPCs)

Question 8

How is Fibrin formed?

Answer 8

Fibrin is a fibrous, non-globular protein involved in the clotting of blood.
It is formed by the action of the protease thrombin on fibrinogen which causes it to polymerize. The polymerized fibrin together with platelets forms a hemostatic plug or clot over a wound site.

Question 9

What is Elastin and what is it composed of?

Answer 9

Major protein component of tissues that requires elasticity like arteries, lungs, bladder, skin and elastic ligaments.

It is composed of soluble tropoelastin protein containing primarily, glycine, valine and modified alanine and proline residues

Question 10

How can elastin/tropoelastin work as a scaffold?

Answer 10

Tropoelastin is a ~65kDa protein that is highly cross-linked to form an insoluble complex.

Question 11

What are CPCs and what do they do?

Answer 11

CPC = Calcium phosphate ceramics are a class of tunable bioactive materials that have been widely used for bone tissue repair and augmentation

They have surface properties that support osteoblast adhesion/proliferation (i.e. osteoconduction) and stimulate new bone formation (i.e. osteoinduction)

More significantly, CPCs have been shown to promote bone growth in vivo, and recruit bone marrow stromal cells (BMSCs) to ectopic sites to induce bone formation

Question 12

What are the features of an ideal extracellular matrix?

Answer 12

3-dimensional
Cross-linked
Porous
Biodegradable
Proper surface chemistry
Matching mechanical strength
Biocompatible
Promotes natural healing
Accessibility
Commercial Feasibility

Question 13

What are some important scaffold variables?

Answer 13

Surface chemistry

Matrix topography

• Cell organization, alignment
• Fiber alignment -> tissue development

Rigidity
5-23 kPa

Porosity

• Large interconnected
• small disconnected

Question 14

Define Mechanotransduction

Answer 14

Conversion of a mechanical stimulus into a biochemical response

Question 15

What are the mechanical forces that may be involved?

Answer 15

Flow-induced shear stress

• Laminar blood flow
• Rhythmic pulses

Uniaxial, Equiaxial stretch

• Magnitude
• Frequency

Question 16

What are the different types of cell culture?

Answer 16

Monolayer (adherent cells)
Suspension (non-adherent cells)
Three-dimensional (scaffolds or templates)

Question 17

What sterilization methods can be used for culturing of cells?

Answer 17

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

Question 18

What are the growth conditions for culturing of cells?

Answer 18

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

Question 19

The use of which type of cell would avoid any potential immunological complications?

Answer 19

Autologous cells

Question 20

Describe the various classifications of cells used for tissue engineering

Answer 20

1. Primary cells
   differentiated cells harvested from the patient (tissue biopsy)
   low cellular yield (can only harvest so much)
   potential age-related problems
2. 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

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

Question 21

What are the two lineages of stem cells?

Answer 21

1. mesenchymal
   give rise to connective tissues (bone, cartilage, etc.)
   although found in some tissues, typically isolated from bone marrow
2. Hematopoietic
   give rise to blood cells and lymphocytes
   isolated from bone marrow, blood (umbilical cord)

Question 22

What does CFU stand for?

Answer 22

Colony-Forming Units

Question 23

Describe the different bioreactors?

Answer 23

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

Question 24

Define a skin graft

Answer 24

A skin graft is a tissue of epidermis and varying amounts of dermis that is detached from its own blood supply and placed in a new area with a new blood supply.

Question 25

Define a skin flap

Answer 25

Any tissue used for reconstruction or wound closure that retains all or part of its original blood supply after the tissue has been moved to the recipient location.

Question 26

What’s the difference between a graft and a flap?

Answer 26

Graft - does NOT maintain original blood supply

Flap - Maintains original blood supply

Question 27

What are the classifications of grafts? (3)

Answer 27

Autografts – A tissue transferred from one part of the body to another.
Homografts/Allograft – tissue transferred from a genetically different individual of the same species.
Xenografts – a graft transferred from an individual of one species to an individual of another species.

Question 28

What are the two types of grafts?

Answer 28

Split Thickness(Partial): Contains 100% of the epidermis and a portion of the dermis. Split thickness grafts are further classified as thin or thick.

Full Thickness: Contains 100% of the epidermis and dermis.