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Pre-clinical Research - Protease Regulation / Modulation

Testing of protease regulation by dressings

Method

Standard MMP solutions are prepared (MMP-2 and/or MMP-9)

Standard sized samples of dressing are placed in a fixed volume of MMP solution (with a known and relevant level of protease activity)

The dressings samples are incubated in the protease solutions under controlled conditions

After incubation, the dressings are removed and the protease solutions tested for protease activity by Gelatin Zymography

The protease activity of MMP solutions exposed to the test dressing is compared to that of fresh (non-exposed) MMP solutions – or to MMP solutions exposed to control (non-protease regulating) dressings (as above)

The level of Protease Regulation is measured in terms of the level of reduction in MMP-associated bleaching of the gelatin zymograms – which is measured with a scanning densitometer

Less bleaching = more protease regulation
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KEY POINTS


     i)   signalling molecules (growth factors).

     ii)  the protease shield – that protects tissues        from damage.

     iii) newly formed wound tissue.





IS YOUR DRESSING A PROTEASE REGULATOR?

gelatin zymography of wound protease levels MMP-9 std MMP-9 std Day 1 Day 3 Day 5 Day 9
Wound fluid Samples

Gelatin Zymography is the preferred technique for the measurement of protease activity by all leading academic laboratories.  

Different proteases have different sizes (molecular weights).

Gel electrophoresis is used to separate proteases within a sample based on their molecular weight.

Gelatin is incorporated within the electrophoresis gels.

Once the proteases are separated by electrophoresis they are activated by the addition of calcium ions.

Activation causes the separated proteases  to digest the gelatin in the electrophoresis gel.

When gels are stained for gelatin, areas where proteases have digested gelatin are translucent (see image above).

The level of protease activity is then determined by measurement of the level of colour loss.

More colour loss = more protease activity      
Testing of wound proteases


Investigation of ‘Protease Regulation’ by dressings

Inactivation/Adsorption of MMPs by dressings

Ion depletion by dressings

Measurement of proteases in clinical trial wound fluid samples

Measurement of proteases in tissue/fluid samples from in vivo studies
protease regulation measured using gelatin zymogram
Control dressing
Protease regulating dressing
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MMP-9