Introduction

Chronic wounds affect an estimated two million people in the United States and are a major socioeconomic burden to the patient and the healthcare system, resulting in an estimated yearly expenditure of upwards of $3 billion.[1] The basic foundation for good wound care involves eliminating exacerbating factors, such as localized pressure in pressure ulcers and diabetic foot wounds and edema in chronic venous stasis ulcers. Standard wound care procedures include wound debridement, the use of dressings to maintain a moist environment, and topical antimicrobial agents when needed to treat wound infections. Although these concepts and regimens improve healing in many wounds, some wounds fail to heal, and this leads to the need to understand the biochemical deficits in the wound microenvironment that prevent wounds from healing.

Acute and chronic wounds have different cytokine, growth factor, proteinase, and mitogenic activity profiles. Fluids obtained from different types of chronic human wounds have elevated levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1b (IL-1b) compared to healing wounds, and elevated matrix metalloproteinases (MMPs) and serine proteases, such as neutrophil elastase.[2–5] Combined with reduced levels of tissue inhibitors of metalloproteinases (TIMPs),[6] the high proteolytic microenvironment in chronic wounds is thought to reduce the ability of chronic wounds to heal by degrading essential growth factors, receptors, and extracellular matrix proteins.[7] For example, studies of specific growth factors in chronic wounds reported reductions in the endogenous levels of TGF-beta and PDGF and a down regulation of PDGF receptor and TGF-beta.[8,9]

Ischemic bipedicle rat skin flap model mimics molecular abnormalities of human chronic wounds. Rapid development of therapies that target specific abnormalities in human chronic wounds is dependent on the use of animal models that mimic the biochemical and structural abnormalities that characterize human chronic wounds. An ischemic bipedicle dorsal rat skin flap model has been reported to have delayed healing and elevated levels of proinflammatory cytokines (TNF-alpha and IL-1beta) and MMPs (pro-MMP-9, active MMP-9, and active MMP-2), similar to those observed in human chronic wounds.[10,11] Thus, this animal model should be useful in evaluating the effects of treatments on altering the microenvironments of wounds and promoting healing. A logical agent to evaluate is PDGF because of its significant role in wound healing. PDGF activates macrophages and fibroblasts and stimulates the production of extracellular matrix proteins and granulation tissue.[12] Also, human recombinant PDGF-BB was shown to increase healing of diabetic foot ulcers and pressure ulcers and is approved by the FDA for the treatment of diabetic foot ulcers.[13–15]

Methods

Bipedicle ischemic skin flap model. Multiple topical applications of human recombinant PDGF-BB and a single application of an adenovirus vector expressing PDGF-BB (Ad-PDGF) were evaluated in the bipedicle ischemic rat skin flap model as described previously.[10,16] Briefly, a 11cm x 2.5cm bipedicle dorsal skin flap centered on the midline between the scapula and iliac crest was raised on the dorsum of 36 adult (250gm) male Sprague Dawley rats. Six full-thickness punch wounds 6mm in diameter were arranged in pairs at 2.6cm, 5.2cm, and 7.8cm from the end of the template and 3mm from each edge, and the skin flap was repositioned and secured by surgical staples.

Topical treatment of wounds with rhPDGF protein. On the day of surgery (day 0) and for the next 13 days, 50µL of either vehicle gel (1% carboxymethylcellulose gel) or 0.01-percent rhPDGF* was applied topically to the wounds of each rat in the two study groups.