When I differentiate osteoblasts from MSCs and treat them with different GAGs, I get normal mineralisation for the untreated cells, but nearly no mineralisation for the treated cells. Funny thing is all markers (ALP activity, Genexpression of Runx2, ALP, Osteocalcin, RANKL/OPG, etc.) that we routinely test for in our lab are not differently regulated.
Proliferation is decreased, but there is no apparent lack in cells in my wellplates. They also seem to produce normal amounts of organic matrix, but simply do not mineralize it even when I extend the cultivation time.
Do you have any ideas what could be disregulated, that is inhibiting the switch from ECM production to ECM mineralization?
Hi Juliane,
you should look for DMP1 expression (dentin matrix protein1) since it has been shown is a key regulator of mineralization. Do you know if there is a delay (longer time in culture might induce mineralization) of just no mineralization?
This first and simplest explanation may be that the GAGs directly block mineralization by binding Ca2+. They are highly negatively charged molecules and could chelate the calcium. Most media have about 2 mM total Calcium. If this is what's going on, the GAGs should have an immediate effect on mineralization. You could grow your cells under differentiating conditions but without beta glycerol phosphate for a long enough time to get high OB marker induction, then add BGP and you will get mineral in a day or so but if you add GAGs with the BGP, they would block mineral if they have a direct effect.
I agree with Renny. GAGs inhibit mineralization unless they are heavily sulfated. This is documented in the older literature. Binding calcium is a very likely explanation. Depending on the GAGs added, release from the matrix of heparin-binding growth factors that could inhibit mineralization is also a consideration.
The above answers seem very likely! Another simple possibility is that the GAGs are reducing the medium pH, osteoblasts will not mineralise in low pH conditions despite producing ample organic matrix. It should be simple enough to check your pH.
How much of cell using for mineralization , you can plate 60 percentage of cells for the treatment groups because your drug may contain proliferative effect that will affect the maturation and mineralization of the osteoblasts and than use the heat inactivated or charcoal striped serum, find out property of your drug first
Thank you all for your detailed answers. I will try different approaches to test wether the chelated calcium is the culprit. The pH is not affected.
To rule out the proliferative effects i already differentiated cells without treatment until there was matrix deposition in a few wells (day 15+). Then i started the GAG treatment and basically got the same results.
They really did chelate the calcium! I increased the Ca to 8mM and got the results fitting to the other data. Thanks again Dr. Franceschi!
Hi everyone, I do mineralize my cell line with 10mM of BGP and 50ug of ascorbic acid. Initially things went well. Due to some problem with the lab condition I cultured cells with 2.5ug of amphotericin and went on with my experiments. Now I'm facing a problem where my cells are not getting minerlization with the conditions I have mentioned above. Does Amphotericin inhibits mineralization of my cell line. If so what should I do now?
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression