Poor healing of DFUs (diabetic foot ulcers) is a major clinical problem that can be extremely debilitating and lead to lower limb amputation. In the normal acute wound, the Cx43 (connexin 43) gap junction protein is down-regulated at the wound edge as a precursor to cell migration and healing. In fibroblasts from the human chronic DFU wound edge there was a striking and significant 10-fold elevation of Cx43 protein, as well as a 6-fold increase in N-cadherin and a 2-fold increase in ZO-1 (zonular occludin-1), compared with unwounded skin. In streptozotocin diabetic rats, Cx43 was found to be up-regulated in intact dermal fibroblasts in direct proportion to blood glucose levels and increased 2-fold further in response to wounding of the skin. To mimic diabetes, NIH 3T3 fibroblasts were cultured under different concentrations of glucose or mannitol and Cx43 protein intercellular communication and migration rates were determined. Cultures of fibroblasts in very high (40 mM) glucose conditions showed significantly elevated Cx43 protein levels, as shown by immunostaining and Western blotting, and significantly increasing gap junctional communication, as shown by dye transfer. In scratch wound-healing assays, increased levels of Cx43 from high glucose resulted in repressed filopodial extensions and significantly slower migration rates than in either standard conditions (5.5 mM glucose) or the osmotic control of mannitol. Conversely, when glucose-induced Cx43 up-regulation was prevented with Cx43shRNA (Cx43 short-hairpin RNA) transduction, the fibroblasts extended long filopodia and migrated significantly faster. Cx43 protein was up-regulated in fibroblasts in DFUs as well as after high glucose exposure in culture which correlated with inhibition of fibroblast migration and is likely to contribute to impaired wound healing.

Scaffolds have been reported to promote healing of hard-to-heal wounds such as burns and chronic ulcers. However, there has been little investigation into the cell biology of wound edge tissues in response to the scaffolds. Here, we assess the impact of collagen scaffolds on mouse full-thickness wound re-epithelialisation during the fi rst 5 days of healing. We fi nd that scaffolds impede wound re-epithelialisation, inducing a bulbous thickening of the wound edge epidermis as opposed to the thin tongue of migratory keratinocytes seen in normal wound healing. Scaffolds also increase the infl ammatory response and the numbers of neutrophils in and around the wound. These effects were also produced by scaffolds made of alginate in the form of fi bers and microspheres, but not as an alginate hydrogel. In addition, we fi nd the gap junction protein connexin 43, which normally down-regulates at the wound edge during re-epithelialisation, to be up-regulated in the bulbous epidermal wound edge. Incorporation of connexin 43 antisense oligodeoxynucleotides into the scaffold can be performed to reduce infl ammation whilst promoting scaffold biocompatibility.

Antisense oligodeoxynucleotides targeting the mRNA of the gap junction protein Cx43 promote tissue repair in a variety of different wounds. Delivery of the antisense drug has most often been achieved by a thermoreversible hydrogel, Pluronic F-127, which is very effective in the short term but does not allow for sustained delivery over several days. For chronic wounds that take a long time to heal, repeated dosing with the drug may be desirable but is not always compatible with conventional treatments such as the weekly changing of compression bandages on venous leg ulcers. Here the coating of collagen scaffolds with antisense oligonucleotides is investigated and a way to provide protection of the oligodeoxynucleotide drug is found in conjunction with sustained release over a 7 d period. This approach significantly reduces the normal foreign body reaction to the scaffold, which induces an increase of Cx43 protein and an inhibition of healing. As a result of the antisense integration into the scaffold, inflammation is reduced with the rate of wound healing and contracture is significantly improved. This coated scaffold approach may be very useful for treating venous leg ulcers and also for providing a sustained release of any other types of oligonucleotide drugs that are being developed.

The gap junction protein connexin 26 (Cx26) is expressed at high levels in naturally hyperthickened epidermal layers as well as pathological hyperkeratotic disease states, such as warts, psoriatic plaques, and chronic wound edges. The overexpression of Cx26 is also linked with inflammation, breakdown of the skin barrier function, and perturbed wound healing. Here, a collagen scaffold implanted into a rat excisional skin wound is used. This induces a foreign body type reaction characterized by epidermal thickening with elevated levels of Cx43 and Cx26, increased inflammation, and perturbed healing. This is reminiscent of a chronic skin wound. If the same scaffolds are coated with an antisense molecule specifically targeting Cx26 that has a slow sustained release, this prevents the abnormal upregulation of Cx26 protein at the wound edge. Knocking down Cx26 protein levels below those seen in normal wound healing has no adverse effects on the healing process but instead reduces the epidermal thickening and also the inflammatory response, while at the same time promotes the healing response. Treatment with Cx43/26 antisense may promote healing of chronic wounds. The Cx26 antisense may also be helpful in treating other skin conditions where Cx26 is overexpressed.