C2-HC8 Flashcards
ECM organization consists of:
ECM organization:
- Core proteins: collagen, proteoglycans, glycoproteins. - Associated proteins: affiliated, secreted factors, regulators.
8 functions of the ECM:
- Anchors: the cell can sense biomechanical factors and this can be redirected via actin, which leads to changes in behaviour.
- Migration barrier
- Migration track
- Signal reservoir: growth factors and cytokines can bind to the ECM, therefore it functions as a reservoir when the cells migrate through the matrix.
- Low affinity co-receptor
- Signal presenter
- Functional fragments
- Biomechanical force
4 properties of the ECM
- The interaction between 2 cell types results in a specific ECM composition.
- ECM influences the endothelial cell behaviour.
- ECM organization is tissue specific
- ECM and cells interact in a dynamic way
Decellularization:
Decellularization= process in which cells and cellular debris are removed from the native or engineered ECM. Therefore, you have a reduced immune response while no cells are present, and you can use it for allogeneic and xenogeneic transplantation. Reduced immune response: allogeneic and xenogeneic transplantation.
Criteria for successful decellularization:
- <50 ng of dsDNA per mg of dry ECM
- Remaining DNA fragments > 200 bp
- Tissue should not have visible nuclear material when stained
The challenge is to retain the mechanical and biochemical cues of the ECM after decellularization.
Balance: remove all the cells vs. Retain the structure of the ECM
Methods for decell.:
Methods for decell.:
- Physical treatment: sonication, pressure, freeze/ thawing.
- Chemical treatment: alkaloids/ acids, detergents (SDS, Tween, Triton), chelators (EDTA).
- Enzymatic treatment: DNAse, trypsin.
In the end you dont see cells via microscopy, but this always needs to be tested with staining.
Efficiency of decellularization depends on:
Efficiency depends on:
- Tissue's cellularity (e.g. Liver vs. Cartilage)
- Density (e.g. Dermis vs. Adipose)
- Lipid content
- Thickness (e.g. Dermis vs. Pericardium)
Problems with partial decellularization:
Problems with partial decellularization: Immune response could affect the regenerative process (cell debris, left over DNA, α-gal epitope). More research needs to be done on the effect of α-gal epitope on the regeneration progress as it does not occur in humans but in pigs and we use pigs to test regenerative methods.
Benefits of decellularized ECM in RM strategies:
Benefits of decellularized ECM in RM strategies: you can decellularize entire tissues and organs that leaves you matrixes and this has a native ECM like composition, but also biomechanical properties and structure, as well as the microstructure in specific parts of the tissue and the entire 3D structure. You can also use in vitro cell culture to get ECM, decellularize it to get the matrix derived and secreted cells. This gives you also a native ECM like composition. It can easily mimic ECM that is difficult to isolate. This gives you more flexibility about modification of the biomaterial, you can knock out genes.
Devitalization:
Devitalization= the process in which cell death is induced, but cellular debris is not removed. It is used as alternative to decell. Why?
- Interesting option for difficult to decellularize tissue (e.g. Cartilage)
- Higher changes of retaining biochemical cues.
Immune response may affect regeneration.
Methods for crosslinking:
Chemical cross-linking
Enzymatic cross-linking
Physical cross-linking (use heat)
Advantages and disadvantages of chemical cross-linking:
Advantages:
- Forming very strong bonds
Disadvantages:
- Almost toxic for cells
- Needs washing to remove the residual cross-linker
- More expensive than physical crosslinking
Advantages and disadvantages of enzymatic cross-linking:
Advantages:
- Unlike many chemical agents, enzymes are most active under mild aqueous reaction conditions - Crosslinking process can often be controlled by modifying temperature, pH, or ionic strength
Disadvantages:
- The most expensive crosslinkers - Substrate specificity
Advantages and disadvantages of physical cross-linking:
Advantages:
- Safe
- Less toxic for cells than chemical agents
- Inexpensive
- Minimum tissue reaction after crosslinking process
Disadvantages:
- Bonds are far weaker than the chemical crosslinkers
- May alter the properties of the materials
- Needs more time for crosslinking
- Lack of control over the reaction kinetics of crosslinking
- Lower degrees of crosslinking
