Laboratory Research
Wound healing is an intricate biological process partially regulated by proteases, including matrix metalloproteinases (MMPs) and serine proteases. Dysregulation of this process results in disruption of the balance in ECM degradation and remodeling, impeding normal wound healing. In this study, we investigated the interaction of Collagen Wound Matrix-Micronized (CWM-MZ*) on common proteases typically found in the wound environment. We also developed an in vitro simulated wound fluid (SWF) model to characterize the impact of CWM-MZ on fibroblasts stressed by SWF. We evaluated the ability of CWM-MZ to rescue normal fibroblast activity including maintenance of cell viability and angiogenic responses to fibroblast eluates.
Methods:
We evaluated the inhibitory effect of CWM-MZ on total protease, collagenase, elastase, and gelatinase activity using gelatin or casein-fluorescein-labeled substrate assays. An in vitro model was developed using transwell inserts with SWF containing a protein-rich salt solution in culture with fibroblasts. Using this model, Collagenase II (50 or 25 CDU/ml) was evaluated alongside CWM-MZ to assess its impact on cell viability (CellTiter-Glo®) after 4 hours of culture at 5% CO2 and 37°C. For tube formation assays, human umbilical vein endothelial (HUVEC) cells were seeded on basement-membrane extract and treated with fibroblast-eluate derived from cells cultured in SWF or SWF +CWM-MZ for 12 hours. Subsequently, the cells were stained with Calcein-AM and imaged.
Results:
CWM-MZ inhibited protease activity by 66%, 45%, and 24%, inhibited collagenases/gelatinases by 80%, 67%, and 50%, and inhibited elastase by 80%, 70%, and 43% at concentrations of 25, 10, and 5 mg/mL, respectively. Using the In-vitro SWF model, we determined that by inhibiting collagenase II activity, the CWM-MZ group preserved cell viability by 95% in the 25 CDU/ml group and 75% in the 50 CDU/ml group. When CWM-MZ was cultured with fibroblasts in SWF, fibroblast eluates resulted in enhanced tube and branch formation of HUVECs compared to SWF alone.
Discussion:
This study demonstrates CWM-MZ's concentration-dependent inhibitory effects on proteases commonly found in chronic wounds. Additionally, the SWF in-vitro model demonstrated how these improvements to the environment may support wound healing responses downstream.