Laboratory Research
Elevated protease activity (EPA) is thought to contribute to wound chronicity by degrading newly formed extracellular matrix proteins in the healing wound. Some advanced wound care biomaterials, mainly collagen-based materials, are thought to provide an alternative substrate for important wound proteases like matrix metalloproteinases (MMPs), potentially protecting newly formed tissue. Other materials, such as keratin, are reported to reduce protease activity in vitro and in vivo, but the mechanism of this is unclear. We investigated if human keratin matrices (HKM) reduced the activity of MMP-1 and -9 collagenases, and if this was due to inactivation or sequestration of the enzymes.
Methods: Solutions of 13.6 U/mL MMP-1 and MMP-9 were incubated with HKM, collagen, or no sample at 37oC for 1 hour in zinc-containing buffer solution (pH 7.5). Supernatants were analyzed for MMP-1 and MMP-9 activity with colorometric assays. Total MMP-1 and MMP-9 levels were also assayed by ELISA. Finally, to evaluate product degradation by MMPs, samples were incubated with the MMP solutions for an additional 23 hours at 37oC, and the change in sample weight over time was calculated. Statistical analysis was performed for each outcome measure with a one-way analysis of variance (ANOVA) with Tukey’s post-test using α=0.05.
Results:
MMP activity was significantly reduced in samples incubated with HKM (p< 0.001) or collagen (p< 0.01). However, total supernatant MMP was reduced in collagen-treated samples (p< 0.05), while there was no difference in protein levels in samples incubated with HKM.
Discussion:
In this work, we found MMP-1 and MMP-9 activity was reduced when incubated with HKM, while total MMP levels remained the same. This suggests the mechanism of MMP activation is not sequestration of the enzymes. Because keratin is reported to be degradation-resistant, these results also suggest HKM is not being preferentially broken down by MMPs, but is instead reducing their proteolytic activity. Though further study is needed into the mechanism by which this occurs, this work shows HKM may benefit chronic wounds by reducing EPA, and also suggests a novel mechanism for enzyme suppression by advanced wound care products.