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9 Cards in this Set

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Describe the kinetics of protein adsorption to your material and how the surface concentration of each protein will change with time.
Due to the Vroman effect, albumin will initially attach, and eventually be replaced by IgM, which has a higher affinity for the material. The higher concentration of the albumin results in a greater initial surface concentration via diffusion, but it will eventually be displaced by the proteins with greater surface affinity (first the transferrin, and finally the IgM).
Subsequently in the experiment, more albumin is added to the synovial fluid. What effect will this have on the proteins adsorbed on the surface?
Addition of albumin, which has less affinity for the material surface, will have minimal effect if IgM is already adsorbed to material surface.
The graph below shows the amount of IgM adsorbed to the material surface as a function of time. What will happen on the material surface with the addition of more IgM?
As can be seen in the graph, a plateau on the amount of IgM adsorbed has not been yet reached, indicating that the surface is not fully covered with the protein. Hence, more IgM will adsorb to the surface.
What determiners of protein structure have the largest impact on protein adsorption to a material? Why?
Dehydration (hydrophobicity), redistribution of charged groups (charge), structural rearrangement (structural stability) of the protein. All these three components play a role in protein adsorption due to their large effect on the Gibbs free energy. Very important !!!
You are performing an in vitro test in which you are testing the adsorption of three
proteins (X, Y, and Z) to a material. After you have exposed the proteins to the material
surface, how do you determine which proteins adsorbed? What assay(s) would be suited
to analyze the solution and the material surface?
You can either look at what is
depleted from the solution, or what is stuck to the material surface. For analyzing the solution: use HPLC or a Western blot assay and examine the remaining solution. Then compare the concentration in the solution to the concentration in an unused control. Whatever is missing should be on the material. For analyzing the material surface: Colorimetric or fluorescent assay.
Some ECM components, such as collagen, can be larger than cells, yet cells are responsible for their formation. How does this occur? Draw a diagram and describe the synthesis of collagen, including all key steps from transcription to formation of fibers in
the extracellular matrix. Provide the location at which each of these steps takes place.
Key steps: Transcription in nucleus Translation on ribosomes in rough ER (mRNA, tRNA, rRNA) Post-translational modification in ER lumen and Golgi Secretion of procollagen to extracellular space cleavage of procollagen chain to allow for assembly into fibrils, fibers and final crosslinking.
How would you test the cytotoxicity of the following materials in vitro? (a) metal for a hip implant (b) degradable scaffold for use in tissue engineering
a) For a metal, you could run a direct contact or agar cytotoxicity assay. b) For a degradable scaffold you could run either one of the previous, but it is also required to run an elution assay, due to the degradation of the material and the potential cytotoxicity of the degradation products.
What is the relative hydrophobicity of the X and Y blocks in the copolymer? Justify your answer based on the data presented above. (graph water angle(polymer composition %X))
X is more hydrophilic. The water contact angle decreases with increasing amount of X.
Explain the dependence of cell attachment on copolymer composition. In particular, provide a justification for the amount of cell attachment observed at 5% X, 25% X, and 100% X. (graph water angle(polymer composition %X))
The data suggest that there is a maximum of cellular attachment on the copolymer that contains 25% of the hydrophilic X. When the percentage of X in the copolymer increases above or decreases below 25%, which in turn increases or decreases its hydrophilicity respectively, cell attachment becomes less favorable. We know that for cells to attach on a surface, protein adsorption to that surface is important, and that cells’ receptors recognize certain binding sites on these proteins. When cellular attachment decreases, we can conclude that either protein adsorption on the material surfaces had decreased or that the proteins have been denatured so that their active sites are not recognizable by the cells’ receptors any more.