Degradation of human collagen isoforms by Clostridium collagenase and the effects of degradation products on cell migration

Abstract

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.

Figure 1

SDS–PAGE showing the degradation of human collagen types I, III, IV, V and VI (U: untreated; T: treated with Clostridium collagenase).

Figure 2

Effect of collagen digests produced by Clostridium collagenase on human keratinocytes migrating on a gold‐coated surface. Collagenase concentration was 5 µg/ml and epidermal growth factor (EGF) at 1 mg/ml was the positive control (*P< 0·05). NT, no treatment.

Figure 3

Effect of collagen (types I and III) digests produced by trypsin (1 mg/ml) on human keratinocytes migrating on a gold‐coated surface (*P< 0·05). NT, no treatment.

Figure 4

Effect of collagen (types I, III and IV) digests produced by Clostridium collagenase on human fibroblast migration. Fibroblast migration to the underside of the membrane was quantified by cell detachment and fluorescent staining, which was detected using the microplate reader. Collagenase concentration was 5 µg/ml and epidermal growth factor (EGF) at 1 mg/ml was the positive control (*P< 0·05). NT, no treatment.