RegeniTherix: Optimised Wound Healing Through Biomarker Detection in a Novel Thermoreversible Hydrogel Dressing

Worsley GJ, Attree SL, Knight AE & Horgan AM

Introduction
According to a recent Department of Health white paper (DH 2010a) the annual budget for the National Health Service stands at over 102bn, and cost efficiency savings are required. Studies indicate that an equivalent of 3% of this total expenditure (roughly 3bn of the annual NHS budget) is required for the care of chronic wounds (Posnett 2007). Unfortunately around 80% of the total cost of chronic wound care is attributed to wound complications and delayed healing, and a reduction in dressing cost would have a limited impact on the cost of care (Drew 2007). Therefore the ability to rationally treat the underlying problems associated with poor or non-healing acute wounds and chronic wounds, that are failing to advance to acute healing wounds, will allow improved clinical management. We present here a wound care system developed as part of the Technology Strategy Board RegeniTherix consortium. One of our aims is to produce a system that will enable prompt medical intervention through the early identification of the underlying causes of delayed wound healing. This will improve patient quality of life and reduce the costs associated with wound complications.

The Smart Dressing Concept

The smart dressing consists of three components (see figure 1):1) A bioresorbable scaffold to be placed directly onto the wound. 2) A thermoreversible hydrogel which will be introduced following application of the scaffold and adsorb analytes via diffusion. 3) A rapid point-of-care device to measure analyte entrapped in the hydrogel.
Figure 2. The RegeniTherix concept. A) The Smart dressing consisting of a bioresorbable scaffold and the thermally reversible hydrogel. B) Point of care testing from the hydrogel trapped analyte.

The Bioresorbable Scaffold

A
The proprietary bioresorbable scaffold developed by our partners at Neotherix has been rationally designed to meet several criteria. In particular the material has been developed to support fibroblast migration and proliferation and to act as a support for the thermoreversible hydrogel. The scanning electron microscope image of the scaffold in Figure 2, gives an example of the effect of fibre diameter on the pore size within the scaffold. These structural characteristics along with the chemical composition of the scaffold were evaluated to select for optimal performance.

Figure 2. Scanning Electron Micrographs of the bioresorbable scaffold demonstrating the tunable nature of fibre diameter and pore size. A) Fibre mean diameter 1.7 m. B) Fibre mean diameter 3.3 m. C) Fibre mean diameter 6.0 m.

The Thermoreversible Hydrogel

The proprietary thermoreversible hydrogel has been specifically formulated so that its lower gelation temperature is beneath skin temperature but higher than normal room temperatures. This will allow the point-of-care practitioner to manipulate the polymer as a liquid solution; once applied directly to the scaffold/wound surface it will heat to above its lower gelation point and form a gel. Gelation of the triblock polymer solution is a physical change brought about by changes in the polymer solubility as the temperature is changed. At temperatures below the Lower Consolute Solution Temperature (LCST) of the poly(lactide-coglycolide) (PLGA) polymer blocks, the triblock copolymer is soluble in water. As the temperature is increased above the LCST, hydrogel bonding between the PLGA blocks and water is disrupted resulting in the polymer becoming increasingly insoluble. Between the PLGA blocks in the triblock copolymer is a region that remains water soluble across the temperature range. The insoluble PLGA blocks are able to form microdomains and bridges between the polymer chains resulting in gel formation (see Figure 3). Because this is a physical process and no permanent chemical changes occur it is fully reversible, and the gel is able to cycle between its liquid and gel state in response to changes in temperature. This feature enables us to release the trapped analyte from the gel for analysis when we revert it back into its solution form.
Figure 3. Representation of a bridged micelle gel network formed by a PLG-PEO-PLGA triblock copolymer above the LCST of the PLGA block. The PLGA block forms insoluble microdomains ( ), surrounded and interconnected by the solvated PEO block ( ).

Point-of-Care Analyser
We have developed a quantitative duplex assay for the detection of wound markers Interleukin-6 and Tumour Necrosis Factor alpha in hydrogel-based samples. The assay is an improvement upon a technique that is becoming a more common point-of-care assay: the Lateral Flow Immunosensor (see Figure 4). With the use of multiple, spectrally discrete, fluorescent microspheres it is possible to quantify the concentration of two or more analytes contained within the gel in a single test. This is achieved through coating multiple capture antibodies within a test-line. The signal from each type of microsphere, which is proportional to the wound marker concentration, is read using a commercially available fluorescence strip reader. Our assays have been validated using real human plasma and hydrogel based samples (see Figure 4 B). The system has a detection limit of 48.5 pg/ml for Interleukin 6 and 55.5 pg/ml for Tumour Necrosis Factor alpha, fulfilling the sensitivity requirement for these wound biomarkers.

Figure 4. A) The Lateral Flow Immunoassay. Sample placed upon the sample pad flows along the strip via capillary action. As the sample moves conjugate beads are resuspended from the conjugate pad and flow along with the sample down the strip. The solution passes the test-line where an immunosandwich is formed if analyte is present. The amount of analyte within the sample is assessed through the intensity of conjugate build up at the test line. Multiple conjugate species if distinguishable can be present within an assay for the detection of different multiple analytes. B) Graphs A and B show dose response curves for Interleukin 6 and TNF alpha using multiplex reagents and hydrogel based samples.

Conclusion
We are developing a smart dressing system that is designed to enable point-of-care practitioners to improve wound management and reduce the cost associated with poor wound healing. This will be achieved through the measurement of wound markers at the bedside and faster clinical intervention.

References
Department of Health (2010) Equity and Excellence. Liberating the NHS. ISBN 9780101788120 Drew P, Posnett J, Rusling L. The cost of wound care for a local population in England. Int J Wound 2007; 4: 149-55. Posnett J, Frank PJ. The costs of skin breakdown and ulceration in the UK. In: Pownall M, ed. Skin Breakdown: The Silent Epidemic. Smith and Nephew Foundation, Hull; 2007: pp 6-12.

Acknowledgments We would like to thank our project partners:Neotherix Ltd SensaPharmc Ltd Complement Genomics Ltd