We were testing these enzymes in our laboratory and were quite surprised how little damage these can do injected into a bovine intervertebral disc organ culture model...
See the following paper for details: Furtwängler T, Chan SC, Bahrenberg G, Richards PJ, Gantenbein-Ritter B (2013) Assessment of the Matrix Degenerative Effects of MMP-3, ADAMTS-4 and HTRA1 injected into a bovine Intervertebral Disc Organ Culture Model. Spine : doi: 10.1097/BRS.0b013e31829ffde8
Interesting work. May be there are factors such as metal ions that need to be made available (i.e. as adjuvants during injection) so the enzymes are more active. Another point is if the possible substrates are enough exposed as to be targeted by the enzymes in the site of injection, may be an enhancement of cell membrane permeability or transport/trafficking is also needed to achieve a degradation via the enzimes injection.
thank you for your comments.
We made sure that the enzymes are activated with pre-incubation mercury containing compound APMA. ADAMTS-4 (R&D) we bought from R&D as activated compound and also MMP-3 (Sigma-Aldrich). We do not know about the distribution of enzymes in the matrix. This is a good point.
TIMP expression was not really up-regulated before and after 7 days of culture as shown with RT-PCR.
What about alpha 2 macro globulin? Is that present. I know it is in serum. What is your readout? We have FRET substrates that you could inject to see if there is any activity.
My guess is that the huge amount of proteglycans present in the fresh bovine discs will bind the positive metal ions which MMPs require, i.e. will deactivate them. Alternatively it can be just not enough penetration of the MMP into a well pressurised disc, so they 'get stuck" surrounded by GAG.
what about mature vs immature? processed vs unprocessed? requirement of cell activation? among all the other mentioned things. Do they work when you just dump them on the 2D cell cultures?
we made sure that we injected activated MMPs, this we considered ;-)
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