Clostridium collagenase has been widely used in biomedical research to dissociate tissues and isolate cells; and, since 1965, as a therapeutic drug for the removal of necrotic wound tissues. Previous studies found that purified collagenase-treated extracellular matrix stimulated cellular response to injury and increased cell proliferation and migration. This article presents an in vitro study investigating the digestive ability of Clostridium collagenase on human collagen types I, III, IV, V and VI. Our results showed that Clostridium collagenase displays proteolytic power to digest all these types of human collagen, except type VI. The degradation products derived were tested in cell migration assays using human keratinocytes (gold surface migration assay) and fibroblasts (chemotaxis cell migration assay). Clostridium collagenase itself and the degradation products of type I and III collagens showed an increase in keratinocyte and fibroblast migration, type IV-induced fibroblast migration only, and the remainder showed no effects compared with the control. The data indicate that Clostridium collagenase can effectively digest collagen isoforms that are present in necrotic wound tissues and suggest that collagenase treatment provides several mechanisms to enhance cell migration: collagenase itself and collagen degradation products.

The data demonstrate that wound pH in a model of contaminated pig wounds is alkaline during the first 10 days of healing. As healing progresses, pH decreases with no significant change in the level of bacterial bioburden. C collagenase exhibited robust activity in the pH range found in this contaminated pig wound model, suggesting it can effectively debride necrotic tissue in the environment found in most chronic wounds in humans.

Small intestine submucosa (SIS), a bioactive extracellular matrix (ECM) containing critical components of the ECM including collagens, proteoglycans, and glycosaminoglycans, has been widely used for wound healing. The purpose of this study was to investigate the interaction between SIS and matrix metalloproteinases (MMPs). MMP-1, MMP-2, and MMP-9 displayed different binding affinities, indicated by a loss in activity in solution upon incubation with SIS at 53·8%, 85·9%, and 36·9% over 24 hours, respectively. A cell migration study was conducted to evaluate the effects of MMPs and SIS on keratinocytes. The results indicated that MMPs inhibit keratinocyte migration in vitro, and that the inhibition can be significantly reduced by pre-incubating the MMP solution with SIS. To evaluate activity in vivo a diabetic mouse wound healing study was conducted. Biopsy samples were collected on different days for analysis of MMP levels by gelatin zymography. MMP activity was found to be attenuated by SIS treatment on day 3 after wounding. On day 7, the attenuation became less significant indicating that the MMP binding ability of SIS had become saturated. SIS was able to reduce MMP activity immediately, and may reduce the inhibitory effects of MMPs on keratinocyte migration.

Antimicrobial dressings are widely used for management of wound bioburden. Frequently, they are used in combination with other topical therapeutic drugs, such as enzymatic debriding agents for the removal of wound necrotic tissues. Such combined applications may have greater potential to achieve multiple healing activities simultaneously, including exudate and bioburden management, debridement, and tissue regeneration. Overall, the authors' testing found that collagenase was observed to be more tolerant when used with the dressings tested than papain. These findings merit further exploration in clinical wounds to confirm clinical validity.

An in vitro efficacy study using newly developed artificial wound eschar (AWE) substrate was conducted for assessing enzyme dose response. The AWE substrate is prepared by the enzymatic conversion of fibrinogen to fibrin in the presence of collagen, fibrin, and elastin to form an insoluble planar matrix. AWE substrate was placed on Franz Diffusion Cells for continuously monitoring the debridement progress. A parallel in vivo study was performed using pig thermal-burn wounds. Papain at concentrations of 200, 400, 800, and 1,600 U/mg was used as the model debriding enzyme for both studies. The data from the first 5 hours of the in vitro testing showed that debriding activity increased as the enzyme concentration increased. The histological results of the in vivo biopsy samples showed that enzyme doses above 800 and 1,600 U/mg successfully achieved debridement on day 8, while lower treatment groups still contained eschar tissue. Using the histological measurement results (wound depth score) a dose response that correlated to the in vitro assessment was found. Granulation tissue maturity and reepithelialization displayed correlation with the enzyme dose. Results indicate that AWE substrate can be used to predict debridement efficacy in vitro when correlation to the in vivo assessment is achieved.