British Journal ojDermatology (1994) 131. 634-640. Gelatinase activity during wound healing M.S.AGREN DeiHirmml of Dertmlobgy and Cutaneous Surgery. University of Miami School of Medicine. PO Box 016250 (R-2S0). Miami. FL iilOl. U.S.A. Accepted for publication 23 March 1994 Summary The two known mammalian gelatinases. 72- and 92-kDa gelatinase. arc extracellular matrix metalioproteinases (MMPs) with a potential role in wound healing. The gelatinase activity as a function of wound age was analysed in tissue extracts of partial- and full-thickness wounds in the skin of pigs, using two assay systems. Total gelatinase activity, assessed using a Hl-labellcd gelatin assay, was highest in the early healing phases and then decreased as healing proceeded in both wound types. Gelatin zymography. which distinguishes the activities ofthe two gelatinases. showed that the 92-kDa (MMP-91 gelatinase essentially followed the same pattern as that of total gelatinase activity, whereas the activity of the 72-kDa gelatinase (MMP-2| remained fairly stable, although it was higher than in uninjured skin, over the experimental period, irrespective of wound type. In conclusion, the two gelatinases appear to have different functions in the wound he;iling process. The 72-kDa gelatinase lMMP-2} is important during the prolonged remodelling phase, whereas the 92- kDa gelatinase (MMP-9) is linked to the epithelialization process and early repair events. Apart from serving as a supporting structure, the extracellular matrix affects cellular activities such as migration, proliferation and protein synthesis. Matrix metalioproteinases (MMPs) are a family of enzymes with at least eight members involved in the metabolism of extracellular matrix proteins. The MMPs are secreted in latent (inactive) forms, which are incapable of proteolysis.^ Latency is main- tained by the linkage between cysteine in the pro- enzyme domain and zinc at the catalytic site. Activation in tissue involves the removal ofthe propep- tide domain, thereby exposing the zinc atom, possibly by the action of plasmin and/or stromelysin. ^ In vitro organomercurials, e.g. aminophenylmercuric acetate (APMA) and sodium dodecyl sulphate fSDS). also activate the enzymes.''' Once activated, another line of control of degradation is exerted by tissue inhibitors of metalioproteinases (TIMP). which bind to the active enzymes with high affinity.' In a healing wound, the extracellular proteins are turned over during the entire repair process. Pre- viously, we studied one MMP. tissue coliagenase (MMP-U. and its activity in different cutaneous wounds in the pig.^ Coliagenase is the only enzyme known to cleave native interstitial collagens (types I. II Correspondence: Dr M.S.Agren, Coloplast A/S. Holtedani 1. DK-J05() Humlcbiek, Denmark. and III) at physiological pH and temperature. Coliage- nase activity was high early after wounding, declined as the wound matured, and returned to the low baseline level of uninjured skin when coverage of the wound with epithelium was achieved. Gelatinases belong to the MMP family, and in vitro studies indicate that coliagenase and gelatinase act in concert when breaking down collagen.^ However, gelatinases degrade not only denatured interstitial collagens further but also native type IV. V and VII collagens. which are present in skin. In a recent study, the two cloned and sequenced 72- and 92-kDa gelatinases. MMP-2 and MMP-9. were examined using substrate gel chromatography before and after making partial- and full-thickness trephine wotinds in the rabbit cornea.'" MMP-2, but not MMP-9. was detected in uninjured cornea. The authors noted different activities ofthe two MMPs as a function of time after wounding, with MMP-9 showing early peak levels, and disappearing after 2 -4 weeks, depending on wound type, whereas MMP-2 levels remained elevated at all time points. In the present study, the sum ofthe activity ofthe two gelatinases was quantified using radiolabeiled gelatin, and MMP-2 and MMP-9 were studied independently and semiquantitatively by substrate gel chromato- graphy in heat extracts of partial- and full-thickness porcine skin wounds. 634