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Advances in Biomaterials > Design Scaffold for Tissue Engineering > Flashcards

Design Scaffold for Tissue Engineering Flashcards

Lecture 2 of 8

1
Q

Describe how a heart is effectively de-cellularised, recellularised and stimulated to pump

A
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2
Q

What different treatment methods are there for decellularisation?

Physical, Chemical and Enzymatic

A

Physical)
Freeze-thaw cycling
Perfusion
Immersion and Agitation

Chemical)
Ionic and Non-ionic detergents
Acids and Bases
Hypertonic and Hypotonic solutions

Enzymatic)
Nucleases
Trypsin

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3
Q

Explain how nucleases are used to decellularise an ECM?

A

Nucleases:

  • Nucleases are enzymes that degrade nucleic acids, including DNA and RNA. These enzymes are used to break down the genetic material of the cells, which helps to eliminate any potential immunogenicity or adverse reactions from the host’s immune system.
  • They do not directly remove the cells themselves from the extracellular matrix (ECM). Instead, nucleases work in conjunction with other agents, such as detergents and mechanical forces, to facilitate the removal of cellular components from the ECM.
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4
Q

Explain how the other enzymatic agent trypsin is used to decellularise an ECM?

A

Trypsin is a proteolytic enzyme that specifically cleaves peptide bonds.
Trypsin treatment helps to dissociate cells from the ECM by disrupting cell-matrix interactions, such as integrin attachments.
The use of trypsin can efficiently strip away cellular components while leaving the ECM scaffold intact, as trypsin primarily targets proteins rather than structural ECM molecules like collagen and elastin.

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5
Q

How do Trypsin and Nucleases work together to decellularise an ECM?

A

Nucleases degrade genetic material, reducing immunogenicity.
Trypsin dissociates cells from ECM.
Combined, they remove cells, preserving ECM for tissue engineering.

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6
Q

Explain how Freeze-thaw cycles decellularise a scaffold

A
  • This treatment subjects tissues to alternating freezing and thawing, a process that disrupts the cellular membranes and results in their removal
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7
Q

Explain how Perfusion decellularises a scaffold?

A

Perfusion involves flushing tissues with solutions containing detergents and enzymes which remove the cells but preserve the ECM

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8
Q

Explain how Immersion and Agitation decellularises a dECM

A

Immersion entails submerging the tissues in decellularising solutions, allowing enzymes and detergents to penetrate the cells.
Agitation applies mechanical force to aid in the removal of these cells enhancing the effectiveness of immersion and perfusion

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9
Q

Explain how Ionic and non-ionic detergents contribute to decellularisation

A

They both disrupt cell membranes, therefore losing their ability to adhere to the ECM and detach

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10
Q

Explain how acids and bases contribute to decellularisation

A
  • Acetic acid and NaOH alter pH, denaturing proteins and also disrupting the cell membranes triggering detachment
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11
Q

How are hypertonic and hypotonic solutions used to decellularise ECMs?

A
  • Hypertonic solutions cause cells to shrink and rupture due to water loss
  • Hypotonic solutions cause cells to swell and burst due to water influx
    Both facilitate lysis and cell detachment
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12
Q

What is Triton-X and how does it work? and what types of ECM should avoid using this treatment?

A
  • Non-ionic detergent
  • It disrupts lipid-lipid and lipid-protein bonds
  • Don’t use to decellularise ECMs with important lipid + GAG components
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13
Q

What is a side effect of using acids/bases to decellularise on the ECM?

A

They can damage collagen and GAG

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14
Q

What is SDS, how does it work and its side effect on ECM

A
  • a non-ionic detergent
  • Its great at denaturing proteins especially those in the cell membrane
  • it can disrupt the proteins of the ECM and leave cell waste in the matrix
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15
Q

What is Triton X-200

A

Similar to Triton X-100
needs to be activated with zwitterionic detergent to be activated

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16
Q

What is CHAPS?

A
  • a detergent with ionic and non-ionic proerties
  • Has similar effect to Triton X-100, disrupts lipid and lipid-protein bonds so dont use on a matrix with high volumes of these
17
Q

What is TBP? And its effect on the ECM?

A
  • It disrupts protein-protein interactions
  • Variable results, it degrades collagen but maintains the mechanical properties?
18
Q

What are EDTA and EGTA? Ant their mechanism to decellularise ECMs

A
  • Ezymatic treatments
  • Break cell adhesion to matrix, and usually combined with Trypsin to remove them from the ECM
19
Q

What is pepsin and its mechanism in decellularising ECM?

A
  • Enzymatic treatment
  • It targets peptide bonds but can cause a lot of damage in the ECM proteins if left for too long
20
Q

What physical methods are available to decellularise an ECM and their effect on the ECM?

A
  • Freezing - disrupts protein structures of the ECM
  • Force - Mechanical can induce cell lysis but this can only be applied to very hard ECMs like bone
  • Agitation - Can damage the ECM
  • Vacuum assisted decellularisation (VAD) - This method just helps facilitate decellularisation by forcing the chemicals into the crevices of the tissue
21
Q

What are some limitations of decellularising and using decellularised ECMs?

A
  • These tissues are in short supply
  • Expensive to produce
  • The decellularization process can subtract from the mechanical and bioactive structure
22
Q

What is going on in this photo?

A
  • Spinach leaf is being decellularised
  • Treated with chemical agents and washed every few days
23
Q

What are some limitations to using plant scaffolds for tissue engineering?

A
  • Residual detergents present after decellularisation can affect cell viability
  • It isnt known how plant vasculature holds up when integrated with human cells
24
Q

Name a few different scaffold types

A

1) Monolithic (single layer)
2) Microporous
3) Nanoparticles
4) Fibrous scaffolds
5) Hydrogels
6) 3D printed

25
Q

What are bulk and surface properties of scaffolds?

A

Bulk - factors that are highly dependent on the volume and quantity of the material

Surface - factors that have low dependence on the quantity and volume of a material

26
Q

List a few bulk properties

A

Porosity/pore size
Interconnectivity
Degradation rate
Mechanical properties
Stiffness

27
Q

List a few surface properties

A

Surface Charge
Topography
Wettability
Biocompatibility

28
Q

How does matrix stiffness affect cells?

A
  • Stiffer matrices result in greater cell adhesion
  • Stiffer matrices also determine the fate of cells, influencing then toward osteogenic lineages
29
Q

What sort of lineage so porous scaffolds and soft scaffolds influence in cells?

A

Porous scaffolds allow the flow of nutrients and waste products promoting the formation of vascular networks and angiogenesis

Soft scaffolds promote differentiation toward chondrocyte lineages

30
Q

How are chondrocytes used to repair cartilage

A

1) Chondrocyte biopsy is taken
2) The chondrocytes are passaged and seeded into a scaffold in-vitro
3) This scaffold is then implanted back into the host to facilitate cartilage repair

31
Q

Explain how traditional tissue engineering works?

A

This method is all done in-vitro
combining scaffold + biological factors + patient derived cells in a lab
The graft scaffold seeded with all elements is then implanted

31
Q

What are the 2 different strategies for tissue engineering?

A

Traditional Tissue Engineering
Modular Tissue Engineering

32
Q

What are some limitations to traditional tissue engineering?

A
  • Complex culture conditions are needed
  • Expensive
  • Risk of immune rejection
  • Poor engraftment efficacy (as the cells aren’t seeded under physiological flow)
33
Q

Explain how modular tissue engineering works

A

This is in-situ tissue regeneration
1) The biomaterial scaffold is loaded with biophysical cues and chemicals
2) This scaffold is then implanted into the body, letting the host do all the cell seeding and culturing in-vivo

34
Q

What are some limitations to the modular tissue engineering apporach

A
  • More difficult to monitor the progress
  • The process is less effective if the implantation site doesnt have young progenitor stem cells that can adapt to form new vascular networks
35
Q

What are some advantages of the modular tissue engineering approach

A
  • This apporach uses the bodys innate regernerative ability
  • improved shelf life of the scaffold
  • lower cost, as intricate seeding and culturing conditions are not needed
  • fewer regulatory hurdles
  • Scalable and provides consistent results and quality

Advances in Biomaterials (9 decks)

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