Hello. I am having some experiment about differentiation periosteal-derived cells to osteoblasts in 2D culture. I cultured cells in OM (osteogenic medium) and additionaly certain amount of BMP-2 was added in medium.
After 7, 14, 28 days I checked ALP activty and calcium deposition. ALP was increased until 14days, but calcium was not detected until 28 days. Plus I did RT-PCR, but late marker (osteocalcin, osteopontin) was not detected.
Has anyone eperienced about osteogenic undifferentiation, please answer.
Thank you for reading and sorry for my bad english.
I dont know the answer, but I recently read this article that talked about it. Perhaps guide you to a solution.
Liu et al. European Journal of Medical Research (2015) 20:10
DOI 10.1186/s40001-015-0084-x
I hope it helps :))
Hi. Can you say what was in your OM? There can be few reasons related to the constitutes of OM eg dexamethasone. in human cells osteocalcin expression is decreased by dex. Did you use any dex? Also, what was the cell density at the start of your experiment?
There are several things to be concerned. Are the cells freshly isolated and had no problem during culture? Some cells detach easily by mechanical stress. And if you lost cells during culture, you won't get much (or any) mineralized nodules. If you didn't see any calcium deposition, it may also difficult to see any upregulated OCN by RT-PCR. In my experiences (but not the same cell type as you have), 7-10 days culture for ALP and 14-20 days for calcium deposition. Additional BMP2 will boost osteogenic differentiation. You can test different concentration (usually 50ng/ml or 100ng/ml).
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