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[anonymous]

How to assay the amount of collagen in cell/scaffold composite?From: ouyangemail: medp9189@nus.edu.sgCategory: Protein

MessageI am doing some research on ligament tissue engineering. I am having some difficulties in assaying the amount of collagen in cells/scaffold composite. Is there anyway I can get the complete protocol for this assay,using radioactive labelling method or other way? hope for your reply!thanks! ouyang email:medp9189@nus.edu.sg

[anonymous]

To determine collagen contents you first have to know if you want to monitor (i) the rate of collagen deposition or (ii) the total amount of collagen.

If you want to determine the total amount of collagen you can simply use a hydroxyproline assay. Look up the method of J.F. Woessner Jr.  For assaying the rate of collagen deposition, you can use a radioassay with tritiated proline. There are several papers dealing with ligament cell cultures (medial collateral, and anterior cruciate) that have used this technique you can look-up as a reference. Remember that proline is incorporated into a number of proteins, so you would have to solubilize the collagen using a specific collagenase first and then do the counts on the supernantant. To quantitate and type the collagens, a reducing PAGE analysis with densitometry measurement of the bands would work as well-a little more work, but it would allow you to determine ratios of collagen types produced by the cells.

You say you are working on ligament engineering? I am also working in this area. I would be interested in hearing more about your project.

Hope this has helped.