you are working with a progenitor cell line to induce fibroblastic differentiati

Question

you are working with a progenitor cell line to induce fibroblastic differentiation on a copolymer of negatively-charged polymer X and neutral-charge polymer Y. You find that your progenitor cells attach and spread in the presence of a new integrin- binding peptide (IBP)

a)You can attach the IBP to the end of two different proteins – one which hasa large negative charge, and one which is highly positively charged. Which protein would you use as a carrier for the IBP and why? 3 sentences

b) You examine water contact angle and progenitor cell spreading on the copolymer surface and find the following results:

Discuss the relative hydrophilicity of polymer X and polymer Y and explain how youarrived at your answer based on the chart above. (3 sentences or less)

c. (10 points) Explain the spreading phenomena observed in terms of surface propertiesof the material and cell and protein interactions with this surface. In particular, provide justification for the amount of cell spreading seen at 5% X, 25% X, and 100% X, as well as why a plateau in cell adhesion is seen at 50-100% X. (5 sentences or less)

d. (10 points) To make sure the results are consistent, you ask a colleague to repeat the experiment above. Your coworker accidentally places all the samples in a mixture containing 75% IBP attached to the negative protein and 25% IBP attached to the positive protein. How would this affect cell adhesion at 5% X and 100% X (give an estimate of % spread cells)? Explain based on protein interactions with the surface and the cells. (6 sentences or less)

(Note: In this case, if all the protein in solution is adsorbed, it will form a single

monolayer on the biomaterial surface.)

e. (5 points) After adhesion, what is one way to check to see if your progenitor cells have differentiated to possess a fibroblastic phenotype? (4 sentences or less)

Spread Cells Polymer Composition Water Contact Angle (deg) 115 18 100 10 35 73 25 85 50 65 92 75 89 40 100 20 93 Spread Cells" refers to the number of cells on the surface that are considered "spread" (e.g. have a surface area larger than a certain threshold value)

Explanation / Answer

c.

d.

a. I will attach the IBP to the positively-charged protein because a positively-charged protein will promote adsorption to negatively charged polymer' X'. The adsorbed IBP will then promote cell adhesion and spreading. b. According to the chart given above, polymer X is more hydrophilic while polymer Y is more hydrophobic. As polymer 'X' composition increases water contact angle decreases which indicate an increase in hydrophilicity.

c.

• We know that IBP is bound to a positively-charged protein so polymer X is negatively-charged so it will promote the adsorption of IBP.
• With increasing percentage composition of polymer X, adsorption of IBP and cell spreading increases.
• The surface carries a little charge, resulting in little IBP adsorption and little spreading at 5% X,
• As % X increases, IBP adsorption, surface negative charge, and spreading increase (as seen at 25% X).
• The cells are highly spread at 100% X.
• Spreading reaches a maximum and plateaus att 50% X and above.
• At 50% and above there is enough negative charge on the surface to attract sufficient IBP to allow cell spreading (100% spreading is not seen, possibly because there is not enough IBP in solution, or not enough space on the surface for enough IBP, to interact with the receptors on all the cells.)

d.

• 5-18% spreading would be expected at 5% X.
• Cell adhesion and spreading will not change much as given in the chart since there is already very little charge on the copolymer, though spreading may decrease slightly.
• Two possible explanations at 100% X:

1. At 100% X, 20-25% spreading may occur because the only one-fourth of the total protein is positively charged and capable of adsorbing to the surface resulting in a similar spread cells (23%).
2. At 100% X, 70-75% spreading may occur because high spreading is observed at 50% X, suggesting that only half of the total protein is required for good adhesion; therefore, one-fourth of the total protein would result in spreading similar to that seen at 25% X (73%).

e. A phenotype can be determined by looking for production of protein markers that are specific to fibroblasts. It can be done using an ELISA or HPLC. Alternatively, the phenotype can be assessed by looking for cell morphology that is consistent with fibroblasts.

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