Wound healing
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2004 – 2007 Dr Hadwen Trust PhD Research Studentship: Development of a chronic wound bioassayDr P Stephens, Prof DW Thomas & Matthew CaleyCardiff University |
For the duration of this project Matthew Caley was a PhD research student funded by the Dr Hadwen Trust. His supervisor was Dr Phil Stephens, a Reader in cell biology at Cardiff University. Dr Stephens established the Wound Biology Group at Cardiff University to investigate the processes which produce and maintain wounds in a variety of diseases.
Impaired wound healing affects 3% of the population over the age of 60, costing the NHS over £1 billion annually and rising. Chronic non-healing wounds such as venous ulcers, diabetic ulcers and pressure sores cause significant distress and disability.
Although numerous animal models of chronic wounds have been developed but none of them accurately reproduces the dysfunctional wound healing responses that occur in older people [1]. In vivo wound models are based on crushing, burns, chemical injuries or induction of tissue ischemia, and can cause considerable pain and distress to the animals involved, which are usually mice, rats, rabbits or guinea pigs.
Despite species- and disease-related differences between animals and humans, animals are increasingly used in the search for new therapies [2]. The development of a robust in vitro model of human wound healing that could be used to screen novel therapeutics would be invaluable for replacing animal tests.
A grant from the Dr Hadwen Trust enabled PhD student Matthew Caley and Dr Phil Stephens to investigate new human cell-based in vitro models of chronic wound healing. The research focused on human fibroblasts, cells known to play an important role in the closure of skin wounds, as they replace and remodel the lost tissue and also influence both re-epithelialisation and angiogenesis.
Building on earlier work studying human fibroblasts from chronic wounds in culture [3], human telomerase (hTERT) immortalised fibroblast cell lines were developed from chronic venous leg ulcers and from patient-matched healthy tissues. These cell lines have an extended life span and a stable genotype and phenotype indistinguishable from primary cells, making them ideal for use in a reproducible in vitro model.
Monolayer scratch wound assays, a basic model of wound repopulation, had shown a reduced rate of wound repopulation in primary chronic wound fibroblasts when compared to normal fibroblasts. This difference was maintained with the immortalised cell lines.
A key aim of this project was to identify genes differentially expressed between wound-derived and healthy fibroblasts, and then to develop reporter cell assays that could be used to replace animals in the assessment of chronic wound treatments.
Microarray and QRT-PCR analysis identified and confirmed a number of disease marker genes that were differentially expressed between chronic wound fibroblasts and healthy fibroblasts, and these alterations were maintained in cell lines. Genes whose activity is modified in wound-derived fibroblasts included those involved in extracellular matrix remodelling, immune system and inflammatory pathways.
A fluorescent protein has been used to generate reporter constructs for two genes of interest and a housekeeping gene. Tests have shown that it is possible to measure changes in fluorescence, and hence gene expression, over time. Further work will involve testing for all the disease genes of interest and to develop stable reporter cell lines for both chronic wound-derived and normal fibroblasts.
The cell lines generated by this project will form the basis of an in vitro chronic wound bioassay for screening novel therapies. The major advantages of such a model of wound healing are that it will be highly reproducible, efficient, easy to use and inexpensive, with strong potential to replace painful tests on animals.
This system may also be seen as proof of principle for the development of other cell lines pertaining to other disease conditions.
Summary
- Immortalised chronic wound-derived and normal fibroblast cell lines have been developed, with a stable genotype and phenotype indistinguishable from primary cells.
- These cell lines have an extended life span, continuing to multiply after 400 days in culture, making them ideal for use in an in vitro model.
- Scratch wound repopulation assays show differences between chronic wound fibroblasts and normal fibroblasts.
- Microarray and QRT-PCR analysis has identified a number of disease marker genes that were differentially expressed between chronic wound fibroblasts and healthy fibroblasts.
- The cell lines generated by this project will form the basis of a reproducible in vitro model chronic wound bioassay for screening novel therapies in place of painful animal tests.
References
- Davidson JM (2001). Experimental animal wound models. Wounds 13:9-23.
- Perez R & Davis SC (2008). Relevance of animal models for wound healing. Wounds 20:3-8.
- Stephens P, Cook H, Hilton J et al (2003). An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype. Exp Cell Res 283:22-35.
- Cook H, Davies KJ, Harding KG et al (2000). Defective extracellular matrix reorganization by chronic wound fibroblasts is associated with alterations in TIMP-1, TIMP-2 and MMP-2 activity. J Invest Dermatol 115:225-233.