Sheets Anthony R, Demidova-Rice Tatiana N, Shi Lei, Ronfard Vincent, Grover Komel V, Herman Ira M
Graduate Program in Cellular & Molecular Physiology, The Sackler School of Graduate Biomedical Sciences, Tufts University, 136 Harrison Ave, Boston, MA, 02111, United States of America.
Department of Developmental, Molecular and Chemical Biology, School of Medicine, Tufts University, 136 Harrison Ave, Boston, MA, 02111, United States of America.
PLoS One. 2016 Jul 26;11(7):e0159598. doi: 10.1371/journal.pone.0159598. eCollection 2016.
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.
清创术，即清除病变、无活力的组织，对于临床医生准确评估伤口状况并为高级治疗或后续的主动愈合准备伤口床至关重要。通过手术或机械方法清除坏死的腐肉和焦痂缺乏特异性，且可能会给患者带来痛苦。酶促清创剂，如梭菌胶原酶，能选择性且无痛地降解失活组织。除了清创活性外，高度纯化的梭菌胶原酶还能积极促进愈合，我们过去的研究表明，用这种酶消化的细胞外基质能产生肽，这些肽在体外可激活细胞对损伤的迁移、增殖和血管生成反应，并在体内促进伤口愈合。有趣的是，尽管胶原酶Santyl®软膏（一种含有梭菌胶原酶和其他非特异性蛋白酶的无菌制剂）是一种广泛认可的酶促清创剂，但其作为主动愈合实体的作用从未得到证实。基于我们之前对纯梭菌胶原酶的研究，我们现在要问，Santyl®中所含的酶混合物是否能产生源自基质的肽，这些肽在体外促进细胞损伤反应并在体内刺激伤口愈合。在这里，我们鉴定出了新的胶原片段，以及源自血小板反应蛋白-1、多聚蛋白-1、纤连蛋白、转化生长因子β诱导蛋白ig-h3和腱生蛋白-C的胶原相关肽，这些肽是由Santyl®胶原酶消化人真皮毛细血管内皮细胞和成纤维细胞基质产生的，它们在体外使细胞增殖和血管生成重塑比对照组增加50 - 100%。使用已建立的愈合受损模型，我们进一步证明，Santyl®软膏中的特定剂量胶原酶以及新鉴定和化学合成的细胞外基质衍生肽比生理盐水处理的对照组显著增加伤口再上皮化60 - 100%。这些结果不仅证实并扩展了我们早期使用纯化胶原酶和基质衍生肽在体外和体内刺激愈合的研究，而且这些由Santyl®产生的、源自基质的肽可能还代表了令人兴奋的新机会，可用于开发通过酶促清创实现的先进伤口愈合疗法，且可能超越清创术本身。
