Figure 1: Elevated levels of senescence associated β-galactosidase activity within prematurely senescent chronic wound fibroblasts 

Currently our research activities are centred on two major areas:

Dysfunctional wound healing in the aged

Chronic wounds are most simply described as wounds that fail to heal.  They occur most frequently on the lower limbs and exist in three principal forms (pressure sores, venous ulcers and diabetic ulcers).  Chronic wounds occur in an estimated 1% of the population and their treatment is currently estimated to cost the NHS over £1 billion/year.  In chronic wounds the normal cellular responses to acute injury are impaired and the wounds are characterised by a defective wound matrix, a failure of re-epithelialisation and prolonged inflammatory response. Through our research efforts we have demonstrated that chronic wound fibroblasts (CWF) are dysfunctional with respect to their normal wound healing functions (proliferation, wound repopulation, extracellular matrix reorganisation; Cook et al., 2000; Stephens et al., 2003a; Stephens et al., 2004) compared to patient matched normal fibroblasts (NF) and that this is related to a significantly reduced proliferative lifespan and early onset of senescence compared to NF (Fig. 1; Wall et al., 2008).  However, investigation of telomere dynamics utilising Single Telomere Length Analysis has indicated that induction of senescence in CWF was telomere-independent.  Microarray and functional analysis has revealed lower expression, production and activity of several CXCL chemokines in CWF compared to NF suggesting that an inability to correctly express a stromal address code is implicated in the disease chronicity (Wall et al., 2008).  

There is increasing evidence to implicate the excessive production of reactive oxygen species (ROS), such as the superoxide radical (O₂^.-) and the highly reactive hydroxyl radical (^.OH) species, by inflammatory cells in the pathogenesis of chronic inflammatory conditions, such as periodontal diseases, rheumatoid arthritis and dermal wounds (Waddington et al., 2000; Moseley et al., 2004b).  Elevated ROS production is also implicated as a causative factor to the ageing process.  Such events result in extensive degradation of extracellular matrices and altered cellular metabolism.  We have discovered that this early onset of cellular senescence is accompanied by an increase in CWF ROS generation, which further contributes to the elevated oxidative stress within the chronic wound environment and further depletes the capacity of CWF to withstand the accumulation of ROS-induced damage (Wall et al., 2008).  We have also demonstrated that there is a lack of major enzymic antioxidant upregulation in CWF suggesting that the adaptive mechanisms of protection against oxidative stress are defective.   This links with our previous identification of a number of oxidative stress-related biomarkers, which we aim to utilise as prognostic/diagnostic biomarkers of chronic wound infection and disease activity (Moseley et al., 2004a & b).  We are currently investigating ways to counteract this oxidative stress imbalance through over expression of antioxidants and novel agents in our cell populations and through the use of biomaterial-based wound dressings.  Other investigations are centred on the use of biopolymers to protect growth factors from the harsh chronic wound environment (Hardwicke et al., 2008)

Our isolation and characterisation of chronic disease cells has driven our efforts to develop a chronic wound reporter cell line as part of our interests in the 3Rs (replacement, reduction and refinement of animals in research).  Cultures of CWF and patient-matched NF cells have been immortalized using a human telomerase containing retroviral vector.  Microarray analysis of the global gene expression profiles of the CWF and NF, both normally and in response to a wounding stimulus, have enabled the identification of disease-specific marker genes.  The promoters of these disease marker genes have been cloned into fluorescent reporter plasmids and transfected into our immortalised cell lines generating an in vitro chronic wound reporter bioassay (Fig. 2).  We envisage, through future generation of this system, that such an in vitro bioassay will be utilized as a high-throughput pre-screening system to study the efficacy of agents to assist dysfunctional wound healing and in so doing replace a large number of animal experiments.

Figure 2: Chronic wound fibroblasts expressing two disease-specific reporter constructs

The Group also has an interest in the microbiology of these wounds and how this can directly and indirectly affect cellular behaviour.  Chronic venous leg ulcer (CVLU) wounds are colonised by a bacterial microflora, even when clinically non-infected and this is most often poly-microbial.  Our cultural analyses have documented that staphylococci, streptococci, enterococci and facultative Gram-negative bacilli are the bacterial groups most frequently recovered from CVLU, with almost 60% of CVLUs shown to harbour anaerobic bacteria such as the peptostreptococci.  Attempts have increasingly been made to investigate both disease association of specific bacterial groups and the relationship between microbial bio-burden and healing in chronic wounds.  To date, no consistent association with healing of these wounds has been demonstrated for any bacterial species.  In some studies it has been suggested that the number of colony forming units of Staphylococcus aureus per gram of tissue may represent a guide to non-healing.  However, this is not uniformly accepted and studies in other polymicrobial chronic infections suggest that specificity of micro-organism is more important than bacterial bio-burden.  We have shown that this is also the case for chronic wounds (Davies et al., 2007).  Furthermore, utilising 16S rRNA and PCR-sequencing as a tool for identifying difficult to culture micro-organisms we have revealed a significantly greater bacterial diversity within these wounds than that revealed by culture alone with the identification novel species of bacteria also (Davies et al., 2001; Hill et al., 2003; Davies et al., 2004).  We have gone on to show in our in vitro systems that these bacteria are detrimental to normal cellular wound healing function (proliferation, migration) through the production of secreted by-products such as short chain fatty acids (Wall et al., 2002; Stephens et al., 2003b).  We are now extending our microbiological investigations through the development and characterisation of wound biofilms within the lab (Fig. 3; Malic et al., 2007) and through an ongoing multi-centre, collaborative investigation centred on the development of a real time reporter dressing for wound infection.  A recent study has shown that chronic wound patients are routinely over-prescribed antibiotics (Howell-Jones et al., 2005) and we are now also investigating the role that this may play in increasing antimicrobial resistance of the wound microflora in this particular patient group. 

Preferential healing within the Oral cavity

Unlike skin wounds, oral mucosal wounds heal with little/no scar formation.  In vitro, we have demonstrated that oral mucosal fibroblasts (OMF) exhibit a distinct phenotype with increased extracellular matrix reorganisational ability, migration and experimental wound repopulation when compared to skin fibroblasts, which is linked to the differential production of proteases and growth factors (Stephens et al., 1996; al-Khateeb et al., 1997; Stephens et al., 2001a & b).  In relation to the lack of scarring of oral mucosal wounds we have also shown that OMF are resistant to transforming growth factor-β₁ driven differentiation to myofibroblasts (Meran et al., 2007; Meran et al., 2008).  We have also demonstrated recently that, compared to patient-matched skin fibroblasts (SF), OMF have a significantly increased lifespan (80-115 populations doublings compared to 40-65 populations doublings for SF) which is linked to the longer telomeres detected within these cells (Enoch et al., 2008). 

 Microarray analysis of OMF and SF has revealed that genes associated with resistance to oxidative stress are upregulated in OMF (Enoch et al., 2008) which in turn is linked to significantly lower ROS generation in OMF versus SF (Lohana et al., 2008).  Indeed, as these antioxidants are known to reduce telomere shortening rates, thereby extending replicative life-span, their increased expression in OMFs may be the reason for their extended proliferative lifespans in vitro and their superior wound healing abilities compared to SF.  Based on these findings, interventional studies, are ongoing in relation to our interest in chronic wounds, involving the retroviral infection of CWF to over-express antioxidants, to determine whether they can delay the onset of oxidative stress-induced, premature senescence in CWF.

We gratefully acknowledge funding for our research from Algipharma (Norway), Astratech (Sweden), Beiersdorf AG (Germany), Convatec Ltd, Diabetes UK, The Engineering and Physical Sciences Research Council, FMC Biopolymer (Norway), The Dr Hadwen Trust, Johnson & Johnson Wound Management, National Institutes of Health (USA), the National Centre for Replacement, Refinement and Reduction of Animals in Research, the Oral and Dental Research Trust, The Osteology Foundation (Switzerland), Research Into Ageing, Royal College of Surgeons (Lon), Royal College of Surgeons (Edin), The Royal Society, Veterans Affairs Medical Center (USA), The Wellcome Trust, Welsh Office for Research and Development for Health and Social Care