In view of these results, it is hypothesized that a specific inhibitor of MMP-9 could potentially be more therapeutically effective than general MMP inhibitors in modulating chronic ulcers towards a healing state.

Chronic venous leg ulcers are a major health issue and represent an often overlooked area of biomedical research. Nevertheless, it is becoming increasingly evident that new approaches to enhance healing outcomes may arise through better understanding the processes involved in the formation of chronic wounds. We have for the first time shown that the terminal purine catabolite uric acid (UA) is elevated in wound fluid (WF) from chronic venous leg ulcers with relative concentrations correlating with wound chronicity. We have also shown a corresponding depletion in UA precursors, including adenosine, with increased wound severity. Further, we have shown that xanthine oxidase, the only enzyme in humans that catalyses the production of UA in conjunction with a burst of free radicals, is active in chronic WF. Taken together, this provides compelling evidence that xanthine oxidase may play a critical role in the formation of chronic wounds by prolonging the inflammatory process.

Chronic ulcers are an important and costly medical issue, imposing considerable pain, reduced mobility and decreased quality of life. The common pathology in these chronic wounds is excessive proteolytic activity, resulting in degradation of key factors critical to the ulcer's ability to heal. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), with many authors suggesting that they need to be inhibited for the ulcer to heal. The studies we report here show that the excessive MMP activity in CWF can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and tethered to a poly(2-hydroxy methacrylate) (PHEMA) hydrogel. Furthermore, these functionalised alendronate hydrogels appear to be biologically inert as assessed in a three-dimensional ex vivo human skin equivalent model. Together, these results highlight the potential use of a tethered MMP inhibitor to inhibit protease activity in wound fluid. This approach may improve wound healing as it still allows MMPs to remain active in the upper cellular layers of the ulcer bed where they perform vital roles in wound healing; thus may offer an attractive new device-orientated wound therapy.

Results obtained indicate that this approach significantly improves separation of proteins present in low concentrations in CWF. This will facilitate the identification of biomarkers in samples collected from patients with ulcers and lead to improved patient therapies and wound care approaches.

Chronic wounds are an important health problem because they are difficult to heal and treatment is often complicated, lengthy and expensive. For a majority of sufferers the most common outcomes are long-term immobility, infection and prolonged hospitalisation. There is therefore an urgent need for effective therapeutics that will enhance ulcer healing and patient quality of life, and will reduce healthcare costs. Studies in our laboratory have revealed elevated levels of purine catabolites in wound fluid from patients with venous leg ulcers. In particular, we have discovered that uric acid is elevated in wound fluid, with higher concentrations correlating with increased wound severity. We have also revealed a corresponding depletion in uric acid precursors, including adenosine. Further, we have revealed that xanthine oxidoreductase, the enzyme that catalyses the production of uric acid, is present at elevated levels in wound fluid. Taken together, these findings provide evidence that xanthine oxidoreductase may have a function in the formation or persistence of chronic wounds. Here we describe the potential function of xanthine oxidoreductase and uric acid accumulation in the wound site, and the effect of xanthine oxidoreductase in potentiating the inflammatory response.

Several different advanced treatments have been used to improve healing in chronic wounds, but none have shown sustained success. The application of topical growth factors (GFs) has displayed some potential, but the varying results, high doses and high costs have limited their widespread adoption. Many treatments have ignored the evidence that wound healing is driven by interactions between extracellular matrix proteins and GFs, not just GFs alone. We report herein that a clinical Good Manufacturing Practice-grade vitronectin:growth factor (cVN:GF) complex is able to stimulate functions relevant to wound repair in vitro, such as enhanced cellular proliferation and migration. Furthermore, we assessed this complex as a topical wound healing agent in a single-arm pilot study using venous leg ulcers, as well as several 'difficult to heal' case studies. The cVN:GF complex was safe and re-epithelialisation was observed in all but 1 of the 30 patients in the pilot study. In addition, the case studies show that this complex may be applied to several ulcer aetiologies, such as venous leg ulcers, diabetic foot ulcers and pressure ulcers. These findings suggest that further evaluation is warranted to determine whether the cVN:GF complex may be an effective topical treatment for chronic wounds.

The biological activity of the insulin-like growth factors (IGF-I and IGF-II) is regulated by six IGF binding proteins (IGFBPs 1-6). To examine the surface of IGF-I that associates with the IGFBPs, we created a series of six IGF-I analogues, [His(4)]-, [Gln(9)]-, [Lys(9)]-, [Ser(16)]-, [Gln(9),Ser(16)]-, and [Lys(9),Ser(16)]IGF-I, that contained substitutions for residues Thr(4), Glu(9), or Phe(16). Substitution of Ser for Phe(16) did not affect secondary structure but significantly decreased the affinity for all IGFBPs by between 14-fold and >330-fold, indicating that Phe(16) is functionally important for IGFBP association. While His(4) or Gln(9) substitutions had little effect on IGFBP affinity, changing the negative charge of Glu(9) to a positive Lys(9) selectively decreased the affinities of IGFBP-2 and -6 by 140- and 30-fold, respectively. Furthermore, the effects of mutations to both residues 9 and 16 appear to be additive. The analogues are biologically active in rat L6 myoblasts and they retain native structure as assessed by their far-UV circular dichroism (CD) profiles. We propose that Phe(16) and adjacent hydrophobic residues (Leu(5) and Leu(54)) form a functional binding pocket for IGFBP association.

Complexes comprised of IGF-I, IGF-binding proteins and the ECM protein vitronectin (VN) stimulate cell migration and growth and can replace the requirement for serum for the ex vivo expansion of cells, as well as promote wound healing in vivo. Moreover, the activity of the complexes is dependent on co-activation of the IGF-I receptor and VN-binding integrins. In view of this we sought to develop chimeric proteins able to recapitulate the action of the multiprotein complex within a single molecular species. We report here the production of two recombinant chimeric proteins, incorporating domains of VN linked to IGF-I, which mimic the functions of the complex. Further, the activity of the chimeric proteins is dependent on co-activation of the IGF-I- and VN-binding cell surface receptors. Clearly the use of chimeras that mimic the activity of growth factor:ECM complexes, such as these, offer manufacturing advantages that ultimately will facilitate translation to cost-effective therapies.