Design Scaffold for Tissue Engineering Flashcards
Part 2
What 3 factors determine the biocompatibility of a material
1) The materials physical and chemical properties
2) The intended use of the material (eg implant site)
3) The elution of chemicals from the device into the body
What is a toxicant?
A chemical that possesses potentially harmful or adverse properties
What factors effect the impact that a toxicant can have?
- The exposure route and site
- The frequency of exposure
- The duration of exposure
What is the dose relationship response?
The degree of response in the host in relation to the amount of material or toxicant present
What does a dose-relationship response look like
How can we test for toxic substances?
Cytotoxicity testing
What is the definition of cytotoxicity?
The level of damage to cells (eg. DNA, Cell perforation…) due to the release of harmful chemicals by the biomaterial or degraded products of the biomaterial
Name the 3 types of quantitative cytotoxicity tests
1) L929 Elution test
2) Direct Contact test
3) Indirect Contact test
What makes a cytotoxic test quantitative rather than qualitative?
The tests provide descriptive categorical data about the effect of the substance on the cells
Explain the L929 Elution test and what it tests for
- It tests for the cytotoxicity of leachable extracts
1) The test material is immersed in a solvent to extract any leachable chemicals
2) L929 mouse fibroblasts are cultured to confluency
3) The cells are exposed to the extract and a control
4) Incubate the cells for 72 hrs
5) Assess cell viability and morphology under microscope
6) Score the degree of cytotoxicity based on a predefined scoring metric
Explain the Direct Contact cytotoxic test and what does it test for?
- It determines if a device material itself is cytotoxic
1) L929 mouse fibroblasts are cultured on a monolayer until confluent
2) Small pieces of test material are placed directly onto the cell monolayer
3) The cells are incubated for 48 hrs
4) Assessment of morphological changes or viability is conducted on exposed cells
5) Score the degree of cytotoxicity based on a predefined score to quantify the results
Explain the Indirect Contact Cytotoxic test and what does it test for?
- It determines if the device itself or material has potential to naturally release toxic chemicals
monolayer until confluent
2) A thin porous layer of agarose (2mm) is placed on top of the cell monolayer, followed by the test material.
3) The cells are incubated for 48 hrs
4) Assessment of morphological changes or viability is conducted on exposed cells
5) Score the degree of cytotoxicity based on a predefined score to quantify the results
What are the 3 Different quantitative cytotoxic tests?
1) Neutral Red Uptake
2) V79 Colony Formation Assay
3) MTT and Related Tests
Describe the Neutral Red Uptake Test and what it is for
- It determines the if a material extract is cytotoxic
This test works by assessing the volume of viable cells with the ability to incorporate and retain red dye within their lysosomes.
1) Red dye is added to the cell culture
2) Healthy cells retain the dye, staining red
3) Since cytotoxic substances disrupt cellular function, dying or dead cells lose their ability to retain the dye
4) After exposure all cells are washed
5) Colorimetric measurement is done to quanitfy amount of red dye that is retained
6) This is compared to a control cell group
Describe the V79 Colony formation Assay and what it is for
- It determines if a material extract is cytotoxic
- This test measures toxicity by evaluating the ability of cells to form colonies after exposure
1) V79 Chinese hamster lung cells are cultured to confluency
2) Cells are exposed to varying concs of test substance
3) After exposure, cells are seeded into new culture dishes and incubated
4) Viable cells proliferate and form colonies
5) Colonies are stained and counted or measured
6) The less or smaller the colonies the more cytotoxic the material
Describe the MTT test and what it is used for
- To determine if a material extract is cytotoxic
- Principle is that viable cells maintain their mitochondrial activity
1) L929 mouse cells are cultured to confluency on many plates
2) The cells are exposed to varying levels of the test substance
3) Yellow MTT solution is added to the plates
4) Metabolic activity converts MTT into purple formazan
5) The absorbance is measured looking at the absorbance of the wavelengths (spectrophotometrically)
What wavelength of light does yellow MTT absorb and purple formazan?
MTT - 720nm
Formazan - 540nm
What are some limitations to cytotoxic tests?
- Generally static and 2D monocultures so they do not replicate the in-vivo conditions
- Done on single cell types, fails to evaluate the effect of multiple cell types working together
What are some advantages of cytotoxic tests?
- The experimental conditions can be strictly controlled and monitored
- There is consistency and reproducibility in the test results
- Can isolate the effects of individual compounds on cell types
Name the different types of pores you can have in scaffolds
1) Closed Pores (red)
2) Open pores (Run through the scaffold)
3) Blind pores (green)
What is the equation to calculate porosity?
What is the equation for total porosity?
Explain how mercury intrusion works
1) The scaffold is submerged in a known volume of mercury
2) Pressure is applied to push the mercury into the scaffold pore networks
3) The volume of mercury infused is measured
What is the equation for interconnection of pores
How do fluid type measurement methods compare to microscopy?
- They have a much larger pore size detection range
- All 1nm-100um
