Processed eggshell membrane powder regulates cellular functions and increase MMP-activity important in early wound healing processes.

Tram T Vuong,Vibeke Høst,Henri-Pierre Suso,Sissel B Rønning,Kristiane Brathagen,Mona E Pedersen,Tamer A E Ahmed,Maxwell T Hincke

doi:10.1371/journal.pone.0201975

Abstract

Avian eggshell membrane (ESM) is a natural biomaterial that has been used as an alternative natural bandage to cure wounds, and is available in large quantities from egg industries. We have previously demonstrated that processed eggshell membrane powder (PEP), aiming to be used in a low cost wound healing product, possesses anti-inflammatory properties. In this study, we further investigated effects of PEP on MMP activities in vitro (a dermal fibroblast cell culture system) and in vivo (a mouse skin wound healing model). Three days incubation with PEP in cell culture led to rearrangement of the actin-cytoskeleton and vinculin in focal adhesions and increased syndecan-4 shedding. In addition, we observed increased matrix metalloproteinase type 2 (MMP-2) enzyme activation, without effects on protein levels of MMP-2 or its regulators (membrane type 1 (MT1)-MMP and tissue inhibitor of matrix metalloproteinase type 2 (TIMP-2). Longer incubation (10 days) led to increased protein levels of MMP-2 and its regulators. We also observed an increased alpha-smooth muscle actin (α-SMA) production, suggesting an effect of PEP on myofibroblast differentiation. In vivo, using the mouse skin wound healing model, PEP treatment (3 days) increased MMP activity at the wound edges, along with increased MMP-2 and MMP-9 protein levels, and increased keratinocyte cell proliferation. Altogether, our data suggest PEP stimulates MMP activity, and with a positive effect on early cellular events during wound healing.

Highlights

Eggshell membrane (ESM) is a promising biological material with a history of use in wound healing
We investigated the effect of Processed eggshell membrane powder (PEP) on the organization of focal adhesions (FA), using immunofluorescent staining for the well-used FA marker vinculin and cytoskeletal F-actin filaments
We investigated the impact of PEP on fibroblast differentiation by culturing fibroblasts with and without PEP for 3 and 10 days, using transforming growth factor- β1 (TGF-β1, 10 ng/ml) as positive control for myofibroblast differentiation

Summary

Introduction

Eggshell membrane (ESM) is a promising biological material with a history of use in wound healing. It has been used as an alternative natural bandage on burned and cut skin injuries for more than four hundred years in Asian countries. Eggshell membrane regulates MMP-activity and cellular activities support in the form of salaries for these authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Biovotec AS contributed with salary for HPS and provided the PEP material used but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author are articulated in the ‘author contributions’ section

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Discussion

Conclusion