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Proteins in Chronic Leg Ulcer Wound Fluid from Patients Undergoing Clinical Hyperbaric Oxygen Therapy - Biomarker Discovery and Validation




A/Professor David Leavesley, PhD1*

A/Professor Terry Walsh, PhD1

Mr James Broadbent, B.App.Sci (Hons1)1


1.Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Queensland., Australia

*Principal Investigator


Hypothesis and Aims




The hypotheses underlying this research project are:


(i)                   Chronic wound fluid (CWF) from patient leg and foot ulcers contains a heterogeneous complement of protein species which may be useful as biological markers or therapeutic targets for wound healing;

(ii)                 Changes in the composition of CWF due to hyperbaric oxygen (HBO) therapy can be detected and monitored throughout longitudinal patient samples using isobaric tags for relative and absolute quantitation (iTRAQ) technology;

(iii)                The temporal presentation of some identified protein species will correlate with wound healing status or HBO treatment number; and

(iv)        Changes in protein presentation related to healing status or HBO treatment number may have a role in the healing of chronic wounds.



Therefore this research project aims to:


(i)                   Examine current findings regarding the composition of wound fluid from chronic ulcers and collate these findings to establish a CWF protein reference list;

(ii)        Observe changes in protein complement in HBO-treated leg ulcer wound fluid and the degree of change for each detected protein in a time- or treatment dependent manner over the course of HBO-encouraged healing using iTRAQ


(ii)                 Examine the relationship between temporal protein presentation and healing

            status /HBO treatment number;

(iv)        Validate target protein presentation (proteins found to have their temporal presentation related to HBO treatment number or healing status) using an unrelated technique, such as Western Blotting, which is generally regarded as the gold standard within the field; and

(v)        Investigate the effect of the identified proteins on the growth and development of skin cells and three-dimensional human skin equivalent composites.


Outcomes and Significance


The major outcomes for this program of research include:

(i)         The summation of data previously describing CWF protein composition in the literature;

(ii)         The identification of novel proteins in CWF;

(iii)        The quantitation of CWF proteins over the course of clinical HBO therapy;

(iv)        The validation of quantitative differences in patient samples using the gold standard technique;

(v)         The association of protein presentation in CWF with ulcer chronicity / HBO treatment regime; and

(vi)        The association of novel proteins present in CWF with chronic ulcer healing.





This project is significant as it will:


  • Be the first to provide an extensive inventory of protein composition in CWF (currently in a draft manuscript);
  • Extend current knowledge of CWF composition through the identification of novel proteins in CWF;
  • Validate the presentation of proteins over the course of HBO-encouraged healing;
  • Be the first to examine CWF composition in a holistic and temporal manner regarding HBO therapy-mediated healing;
  • Establish new workflows which will be directly applicable to other temporal chronic wound healing investigations, such as compression bandage-mediated venous ulcer healing;
  • Provide clear investigative leads to the molecular mechanisms of HBO-mediated wound healing; and
  • Help to evaluate HBO therapy for the healing of chronic wounds through the generation of quantitative scientific data.


14 January 2009







Improved ex vivo skin models for the evaluation of hyperbaric oxygen-mediated wound healing.




Ms Rebecca Dawson1* (M.Med.Sci)

Dr Jos Malda1,2 (MSc, PhD)


1. Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology, Kelvin Grove, Queensland, Australia.

2. Department of Orthopaedics, University Medical Centre Utrecht, Utrecht, Netherlands.

*Principal Investigator


Hypothesis and Aims


The goal of this project is to further refine and validate our multicellular, 3-dimensional (3D) ex vivo human skin equivalent (HSE) model for the evaluation of hyperbaric oxygen therapy (HBO)-mediated wound healing.


We hypothesise that adding the different types of cells found in vivo skin to the HSE model will lead to a more physiologically relevant model for assessing HBO-mediated wound therapies. Further, this will avoid the need to move to pre-clinical studies in large animals in early stage R&D. To investigate this hypothesis we will pursue the following aims:


Aim 1: To construct a 3D skin model that shares greater similarity to in vivo skin, by incorporating not only the keratinocytes and fibroblasts, but also other cells types found in vivo. These additional cell types will include microvascullar endothelial cells (MVEC), melanocytes and Langerhan cells.


Aim 2: To construct 3D HSE models as in Aim 1 but using cells derived from skin samples taken from the periphery  of chronic wounds.


Aim 3: To compare the responses of the models to HBO treatments using a laboratory scale HBO unit.


Outcomes and Significance


Chronic leg ulcers cost the Australian health care system an estimated $2.6 Billion/year. The condition is difficult to heal, commonly becomes a long-term problem and is increasingly prevalent in the ageing Australian population. It is proposed that effective treatment can be achieved through use of hyperbaric oxygen (HBO) therapy; however, further  scientific evidence is required. Data generated from the experiments described in this application will provide key information addressing the relationship between each cell type present in the 3D HSE resulting in a next generation, improved model suitable for fundamental R&D studies and preclinical testing. The HSE will mimic the behaviour of skin in vivo more closely and will be used for testing wound healing therapies, especially HBO therapy. Therefore, this project will develop a novel in vitro 3D human skin equivalent model to advance the fundamental knowledge base of the discipline and provide further insights into the effect of HBO treatment regimes on wound healing.


14 January 2009




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