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Pharmacology of Regenerative Medicine > Tissue Engineering > Flashcards

Tissue Engineering Flashcards

1
Q

What are the five basic concepts of tissue engineering?

A

Tissue engineering is very bespoke and specific for one patient.

Step 1: Isolate the patients one cells via an appropraite sampling process: results in a mixed population of cells - don’t get lucky enough to get one cell type.

Step 2: Then we need to purify/enrich the desired cell types and expand them in cell culture.

Step 3: Seed them onto an appropriate scaffold.

Step 4: Mature them in a bioreactor that has the right growth conditions for the cell type desired.

Step 5: Implantation

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2
Q

What are the two different cell sources for tissue engineering?

A

Autogenic: Own body: MSCs, iPS cells, Satellite cells, Differentiated cells.

Allogenic: Someone else: often immune response: ES cells, iPS cells, MSCs, differentiated cells.

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3
Q

Biopsies and aspirates may contain numerous cell types, so the cell type of interest needs to be purified, removing unwanted cell types. What are 4 different techniques for this?

A

Differential adhesion: some cells will adhere to certain
surfaces.

Density centrifugation: sort by cell size.

FACS: sorts by size, granularity, surface markers.

MACS: sorts uses magnetic fields based on cell surface markers.

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4
Q

What is the importance of collagen/gelatin balls used in cell isolated and expansion?

A

They are needed so that the cells have a surface to adhere to and grow in suspension - they won’t otherwise.

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5
Q

What are the ideal properties of 3D scaffolds for cell growth? [7]

A
  1. Biocompatible with human body - no allergic reactions.
  2. Biodegradable - eventually degrade in the body to non-harmful constituents, leaving healthy tissue behind.
  3. Cytocompatible - compatible with cells.
  4. Porous - no porosity -> no nutrients to middle of scaffold and no waste out, dead cell mass.
  5. Mechanically appropriate - need to match the mechanic features of tissue going into.
  6. Architecturally appropriate.
  7. Growth promoting - controlled drug/GF release.
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6
Q

Scaffold materials include polypeptides and polysaccharides. What are examples of both?

A

PP: collagen, gelatin, fibronectin, fibrin, laminin, silk fibroin.

PS: Hyaluronic acid, Alginate, Chitosan

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7
Q

PP used as scaffolds [6]

A

Collagen, gelatin, fibronectin, fibrin, laminin, silk fibroin.

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8
Q

PS used as scaffolds: [3]

A

Hyaluronic acid,

Alginate, Chitosan

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9
Q

What is the rationale behind using synthetic polymers as scaffold materials? [4]

A
  1. Control of the degradation, strength, chemical functionality and biological signals.
  2. Reproducibility.
  3. Bulk production.
  4. Interesting properties: temperature responsive release of contents etc.

Examples: Poly(caprolactone), Poly(lactic acid), Poly(glycolic acid), Poly(lactic-co-glycolic acid) PLGA.

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10
Q

Poly(caprolactone), Poly(lactic acid), Poly(glycolic acid) and Poly(lactic-co-glycolic acid) are examples of what type of scaffold?

A

Polyesters

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11
Q

4 examples of polyesters used as scaffolds:

A

Poly(caprolactone), Poly(lactic acid), Poly(glycolic acid) and Poly(lactic-co-glycolic acid) are examples of what type of scaffold?

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12
Q

What are the 5 main different materials scaffolds for tissue engineering can be made from?

A
  1. polypeptides
  2. polysaccharides
  3. synthetic polymers
  4. Bioceramics and bioactive glasses.
  5. Decellularised tissues.
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13
Q

What is decellularisation and how does it relate to tissue engineering?

A

Decellularization is the process used in biomedical engineering to isolate the extracellular matrix (ECM) of a tissue from its inhabiting cells, leaving an ECM scaffold of the original tissue, which can be used in artificial organ and tissue regeneration.

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14
Q

Why was decellularisation developed?

A

This process creates a natural biomaterial to act as a scaffold for cell growth, differentiation and tissue development. By recellularizing an ECM scaffold with a patient’s own cells, the adverse immune response is eliminated.

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15
Q

Bioactive glasses are great scaffolds for the development of

A

Bone-like matrixes.

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16
Q

What are bioactive glasses composed of?

A

SiO2, Na2O, CaO and P2O5.

17
Q

What are some of the methods of scaffold formation? (not hydrogels)

A 
Compression
Solvent casting
Particle leaching
Freeze drying 
Spinning - wet, dry and melt. 
Electrospinning 
3D spinning.
18
Q

The formation of hydrogels for scaffolds is driven by a number of different mechanisms, depending on the polymer, for example:

A 
Thermal 
Ionic
UV
Enzymatic
Covalent
19
Q

What modifications typically need to be applied to scaffolds to enable them to be cell adhesive or growth promoting?

A

Scaffolds may require certain motifs to quide cell function and fate.

This may include the immobilisation of protein, enzyme, growth factors, drugs etc onto the functionalised surfaces of the scaffolds following plasma or wet chemical treatment.

20
Q

________, in particular, can be readily modified using NaOH or primary amines.

A

Polyesters, in particular, can be readily modified using NaOH or primary amines

Subsequent reaction with coupling reagents can be used to attach bioactive motifs (or whole proteins)

Potential for scaffold patterning

21
Q

Polyesters, in particular, can be readily modified using ____ or primary ______.

Subsequent reaction with coupling reagents can be used to attach bioactive motifs (or whole proteins)

Potential for scaffold patterning

A

Polyesters, in particular, can be readily modified using NaOH or primary amines

Subsequent reaction with coupling reagents can be used to attach bioactive motifs (or whole proteins)

Potential for scaffold patterning

22
Q

What is RGD?

A

Arg-Gly-Asp - attached to fibronectin, laminin, collagen, vitronectin = increased cells. `

23
Q

Nerve cells love to grow on what

A

Laminins

24
Q

Why is scaffold morphology important in tissue engineering?

A

Cells do not live in the 2D environment provided by many scaffolds: the tissue environment is 3D.

25
Q

What are the morphological features of collagen-coated glass? [6]

A
  1. Soluble gradients are absent.
  2. Forced apical-basal polarity is present.
  3. Continuous layer of matrix is present.
  4. Unconstrained spreading and migration in x-y directions.
  5. Adhesions restricted to x-y plane.
  6. High stiffness (GPa range)
26
Q

What are the morphological features of collagen gel (3D)? [6]

A
  1. Soluble gradients are present
  2. No presribed polarity
  3. Discrete matrix fibrils
  4. Spreading and migration is sterically hindered - as in the body.
  5. Adhesons are distributed in all three dimensions
  6. Low stiffness (kPA range)
27
Q

Although hydrogel scaffolds provide 3D signalling, and the ability of cells to recieve signals in three dimensions is important, why may hydrogels not be appropriate?

A

Due to the mechanical demands of the tissue being repaired.

28
Q

What is electrospinning?

A

Invovles a syringe driver, a polymer solution and a high voltage power supply.

29
Q

How does electrospinning function, given that it involves a syringe driver, polymer solution and high voltage power supply?

A

When a pendant droplet is formed at the spinneret, an electric voltage bias (up to 100 kV, most often of 10-30 kV) is applied between the tip and a collector placed in front of it, at a distance from a few cm to a few tens of cm. The bias is applied by means of a high-voltage generator. The applied voltage is gradually increased, the droplet elongates according forming an apex (Taylor cone), and finally electric forces overcome surface tension and a jet is produced. The velocity of the jet can reach values of a few m/s, and strain rates are up to 107 s-1.

30
Q

What is a taylor cone?

A

When a pendant droplet is formed at the spinneret, an electric voltage bias (up to 100 kV, most often of 10-30 kV) is applied between the tip and a collector placed in front of it, at a distance from a few cm to a few tens of cm. The bias is applied by means of a high-voltage generator. The applied voltage is gradually increased, the droplet elongates according forming an apex (Taylor cone), and finally electric forces overcome surface tension and a jet is produced. The velocity of the jet can reach values of a few m/s, and strain rates are up to 107 s-1.

31
Q

The dimensions of the nanofibres produced by electrospinning are similar to what?

A

That of the ECM.

32
Q

What conditions of electrospinning can be varied?

A
  1. Distance from needle to plate.
  2. Composition of polymer mix.
  3. The speed at which the plate rotates.
  4. The voltage applied.
33
Q

How do physical and chemical cues affect stem cell fate?

A

The best patterning is disordered square for OPN and OCN. Better than hexgonal, square and random.

Geometric: The expression of BM MSCs on fibronectin patterns differes between different geometric shapes.

34
Q

What is the purpose of bioreactors?

A

They allow cells to be seeded and matured in vitro, while supplying the different physical and chemical cues that may be required for each cell type: O2 concentration, fluid flow, forces.

35
Q

What are the 7 different types of bioreactors availabe?

A
  1. Spinner flask
  2. Rotary Cell Culture
  3. Tension reactor
  4. Perfusion reactor
  5. Compression
  6. Fluidised bed
  7. Hollow fibre bioreactors
36
Q

Which of the following is better for the growth of cartillage?
Static bioreactor
Spinner-flask
Rotating well culture.

A

The rotating well culture.

37
Q

What is used as a model in tissue engineering studies due to its difficulty to generate in vitro?

A

The temporomandibular joint (TMJ).

Clinical relevance: ~25% of the population exhibit signs of TMJ disorders.

38
Q

How has the TMJ been replicated?

A

Via a combination of natural scaffold, bioreactor technology and cell biology.

Pharmacology of Regenerative Medicine (14 decks)

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