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Wound Healing Models Physical Testing Protease Regulation / Modulation Anti-Microbial Efficacy Testing Haemostat Assessment Bio-compatibilty / degradation

•	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

KEY POINTS

• Excessive activity of proteases is thought to be central to the development and persistence of chronic wounds.

• Proteases damage:

     i)   signalling molecules (growth factors).

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

     iii) newly formed wound tissue.

• Various proteases are found in wounds including Matrix Metallo-Proteases (MMPs) which are known to play a central role in wound chronicity.

• Supporting evidence for claiming protease regulatory status of products

• Dressings that can regulate protease activity may encourage chronic wound healing

IS YOUR DRESSING A PROTEASE REGULATOR?

•	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

•	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

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