Novel clay gels as regenerative microenvironments for the treatment of diabetic foot ulcers

Abstract

People that suffer with diabetes have an elevated risk of developing diabetic foot ulcers (DFUs), a type of chronic wound associated with hyperglycaemia. Current DFU treatments vary in their effectiveness and people that suffer with DFUs have an increased risk of eventual lower limb amputation. Biological agents including vascular endothelial growth factors (VEGF) and agonists of the Wnt signalling pathway have been investigated as agents that improve wound healing, yet delivery in an active economical form remains a significant clinical challenge. In this project, we tested the hypothesis that Laponite, a synthetic smectite clay biomaterial, can be used to localise bioactive molecules to skin injury sites and can increase the rate and quality of wound healing. Growth factor (VEGF) and small molecule (BIO, a Wnt pathway agonist) were incorporated in Laponite hydrogels, and their activity tested using cell-reporter assays. Laponite hydrogels were then administered subcutaneously and applied to circular wounds made on the dorsal skin of healthy and diabetic (db/db) mice. Bioactivity of localised VEGF by Laponite gels and its effect on wound healing was assessed by measuring the rate of wound closure and by histological analysis of blood vessel formation, the degree of cell invasion and the rate of re-epithelisation. Human vein endothelial cell (HUVEC) tube formation was significantly increased when cultured on Laponite gels premixed with 1-5 µg/ml VEGF (p = < 0.05-< 0.0001). Successful adsorption of BIO by Laponite from aqueous media was measured at early time points. There was substantial retention of premixed BIO by Laponite over 7 days with the only evidence of BIO release (4.76%) into aqueous media measured at 24 hours (p = < 0.01). Laponite hydrogels were successfully retained in vivo up to 21 days (subcutaneously) and 18 days (wounds). There was a significant increase in blood vessel ingrowth into subcutaneously injected Laponite gels premixed with 10-40 µg/ml VEGF after 21 days in healthy mice (p = < 0.05-< 0.0001). Treatment of back skin wounds of db/db mice with vehicle Laponite gels exhibited a significant increase in the rate of re-epithelisation (p = < 0.05) and improvement to wound healing quality both visually and histologically. No further improvements to the rate of wound closure, re-epithelisation and appearance (e.g. reduced redness) was present when treated with Laponite gels containing 40 µg/ml VEGF; 1.1% alginate gel controls containing 40 µg/ml VEGF did exhibit improved wound closure suggesting that more rapid release of VEGF was more appropriate to a wound model that requires < 30 days recovery. These results show the potential of Laponite hydrogels localising biologically active molecules to skin injury sites and improving the rate and quality of wound healing. However additional in vitro analysis and a modified murine model that would better characterise impaired wound healing in human DFUs, would be required to explore these findings in greater detail.

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ORCID for Nicholas Evans: orcid.org/0000-0002-3255-4388

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