Venous leg ulcers (VLU) represent a clinical challenge and impair the patient’s quality of life. Venous insufficiency is underlying the protracted healing. We report the protein expression pattern for biomarkers in VLU wound fluids and compare these with acute split-thickness wound fluids. Fifty-seven patients with VLU were recruited and treated for 12 weeks with a protease-modulating polyacrylate wound dressing and a two-layer bandage compression system. Ten patients with acute wounds were followed for 21 days. Wound size decreased from 18.7 ± 12.3 cm2 to 11.75 ± 11.39 cm2 in the VLU cohort, 10 wounds healed completely. Relative wound area reduction reached 48.9% ± 51.9% at week 12. 61.4% of the patients achieved a relative wound area reduction of ≥40% and 50.9% a relative wound area reduction of ≥60%. IL-1beta, TNF-alpha, and MMP9 concentration ranges were similar in VLU and acute wound fluids. Concentrations of S100A8, S100A9, neutrophil elastase, MMP2, and fibronectin were elevated in VLU wound fluids and decreased significantly in abundance after two weeks of treatment. The values remained low, yet, higher than those of acute wounds. Collagen (I) alpha1 abundance was higher in VLU wound fluids and not significantly regulated. Our data show a robust healing response in venous leg ulcers treated with a protease-modulating polyacrylate wound dressing. The biomarker pattern in VLU wound fluids changed within the first two weeks and we speculate that this might reflect a decrease of STAT3 influence in the wound bed.

A total of 100 µg protein were denatured in 2.5 M guanidine hydrochloride (GuHCl), 100 mM HEPES pH 7.8, cysteines reduced with tris(2-carboxyethyl)phosphine and alkylated with chloroacetamide, followed by tryptic digest (1:50 protease: protein (w:w)) overnight after adjusting the GuHCl concentration to 0.5 M. Samples (1000 ng of sample peptides and 0.8 pmol of heavy peptides) were loaded on EvoTip columns and separated on an Evosep One chromatography system coupled in line with an Exploris 480 mass spectrometer (Thermo Fisher Scientific). Separation was achieved from the stationary phase over 11.5 min according to the manufacturer standard method “100 samples per day” (100SPD) and the SureQuant(TM) analysis mode containing inclusion lists of peptide precursors and their transitions was applied to record the spectra. Peak processing and filtering were performed in Skyline 20.1. The processed report containing area under the curve (AUC) values for the precursor transitions selected was exported for further analysis. The total ion current chromatogram areas for all MS runs were extracted from the raw data and used to normalize the transition AUC values. Protein values were calculated by the sum of peptide transition values, with two peptides quantified for each protein. Protein values were rescaled for each protein separately to a 0-100 scale for comparison and visualization.

For all patients, baseline exudate was sampled by the application of a sterile non-woven compress (Medicomp® sterile, 70% cellulose fiber, 30% polyester fiber; PAUL HARTMANN AG, Heidenheim, Germany) for two hours before any debridement or wound cleansing occurred, followed by application of a HydroClean® dressing. The soaked Medicomp® compress was put in plastic bag and frozen at -20°C until the extraction steps. For all other time points, the polyacrylate-containing dressings (HydroClean®, PAUL HARTMANN AG, Heidenheim, Germany) were removed at the visits, transferred into plastic bags, and frozen at -20oC until biochemical extraction. The soaked Medicomp® (entire dressing) and Hydroclean® (inner polyacrylate layer) dressings were incubated in a buffer solution with protease inhibitor cocktail to extract the proteins. Polymer residues were removed by filtering the liquid phase through a 40 µL cell strainer (Fisherbrand™ Sterile Cell Strainers, 22-363-547, Fischer Scientific), and cell debris removed by centrifugation (3x 15 min, 4000 g, 4°C) followed by sample concentrating (3 kDa Amicon® Ultra-15 Centrifugal Filter Unit, UFC9003, Millipore, Merck).