Identification and Characterization of Novel Matrix-Derived Bioactive Peptides: A Role for Collagenase from Santyl® Ointment in Post-Debridement Wound Healing?

Anthony R Sheets,Vincent Ronfard,Tatiana N Demidova-Rice,Lei Shi,Komel V Grover,Ira M Herman,Che John Connon

doi:10.1371/journal.pone.0159598

Abstract

Debridement, the removal of diseased, nonviable tissue, is critical for clinicians to readily assess wound status and prepare the wound bed for advanced therapeutics or downstream active healing. Removing necrotic slough and eschar through surgical or mechanical methods is less specific and may be painful for patients. Enzymatic debridement agents, such as Clostridial collagenase, selectively and painlessly degrade devitalized tissue. In addition to its debriding activities, highly-purified Clostridial collagenase actively promotes healing, and our past studies reveal that extracellular matrices digested with this enzyme yield peptides that activate cellular migratory, proliferative and angiogenic responses to injury in vitro, and promote wound closure in vivo. Intriguingly, while collagenase Santyl® ointment, a sterile preparation containing Clostridial collagenases and other non-specific proteases, is a well-accepted enzymatic debridement agent, its role as an active healing entity has never been established. Based on our previous studies of pure Clostridial collagenase, we now ask whether the mixture of enzymes contained within Santyl® produces matrix-derived peptides that promote cellular injury responses in vitro and stimulate wound closure in vivo. Here, we identify novel collagen fragments, along with collagen-associated peptides derived from thrombospondin-1, multimerin-1, fibronectin, TGFβ-induced protein ig-h3 and tenascin-C, generated from Santyl® collagenase-digested human dermal capillary endothelial and fibroblastic matrices, which increase cell proliferation and angiogenic remodeling in vitro by 50–100% over controls. Using an established model of impaired healing, we further demonstrate a specific dose of collagenase from Santyl® ointment, as well as the newly-identified and chemically-synthesized ECM-derived peptides significantly increase wound re-epithelialization by 60–100% over saline-treated controls. These results not only confirm and extend our earlier studies using purified collagenase- and matrix-derived peptides to stimulate healing in vitro and in vivo, but these Santyl®-generated, matrix-derived peptides may also represent exciting new opportunities for creating advanced wound healing therapies that are enabled by enzymatic debridement and potentially go beyond debridement.

Highlights

Acute cutaneous wound healing occurs through a well-orchestrated series of events that culminate in tissue repair and wound closure [1]
Since our previous studies indicate that pure Clostridial collagenase significantly promotes cell migration and proliferation in vitro and stimulates wound healing in vivo [25] through liberation of bioactive, matrix-derived peptides [26,27], we aimed to reveal whether Santyl1 collagenase, through its proven and selective debridement activity, liberates specific collagen- and collagen-associated peptides that promote cellular responses to injury, including cellular growth and capillary endothelial cell-driven angiogenesis in vitro; and, if so, whether such bioactive peptides could further induce wound-healing angiogenesis and re-epithelialization in murine models of impaired healing [27]
To determine the identity of the extracellular matrix (ECM)-derived molecular species released during Santyl1 collagenase-mediated ECM digestion, liquid chromatography-tandem mass spectroscopy was performed on select sodium dodecyl sulfate (SDS) polyacrylamide gel-electrophoresed protein bands of interest and present in the collagenase-digested but not control treated ECM preparations

Summary

Introduction

Acute cutaneous wound healing occurs through a well-orchestrated series of events that culminate in tissue repair and wound closure [1]. Matrix-integrin interactions dictate growth and migration of keratinocytes, fibroblasts, and vascular endothelial cells, control morphogenic processes such as endothelial tube formation during angiogenesis, and determine synthesis of additional matrix components [3,6]. In response to matrix-derived and injury-provoked events, dermal and epidermal cells, alike, further modify their respective extracellular matrix microenvironments, often giving rise to matrix remodeling [3,8]. Such changes in matrix architecture feedback to the cells and provide additional instruction, perpetuating the cycle of dynamic and reciprocal interactions, known as dynamic reciprocity, between cells and their surrounding ECM that are critical for wound repair [3]

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