EUROPEAN  TISSUE  REPAIR  SOCIETY

THE THERAPEUTIC USE OF GROWTH FACTORS IN CLINICAL PRACTICE
Martin C. Robson, Professor Emeritus of Surgery, University of South Florida

Cytokine growth factors are mediators of the multiple cellular processes of the wound healing scheme. With the advent of DNA technology and the mapping of the human genome, growth factors are available for study in clinical trials. To date, several growth factors have been applied topically to chronic wounds with variable results. One factor, rhPDGF-BB has been approved for the treatment of diabetic foot ulcers.
The data for the various clinical trials can be divided into the three most common chronic wounds, the diabetic neuropathic foot ulcer, the pressure ulcer and the venous stasis ulcer. Four trials were reported for PDGF (Regranex) for the treatment of diabetic foot ulcers. At the ETRS meeting in Bordeaux, TGF-beta 2 was demonstrated to have effects comparable to PDGF. Basic FGF (bFGF) has been tried in diabetic foot ulcers without success.
PDGF has had partial success in three reported pressure ulcer trials. Problems in trial design have prevented a statistically acceptable beneficial effect being demonstrated in this wound. BFGF has also been demonstrated to have a beneficial effect in pressure ulcers. Recently, it has been shown to yield better long term healing rates than other modalities including surgery.
Three growth factors have had some success in accelerating healing of venous stasis ulcers. TGF-beta2 was reported in two trials to increase healing rates. At the World Congress for Wound Healing in Melbourne, KGF-2 (Repi-fermin) showed very promising results in a 12-week trial. A longer trial of over 600 patients is now underway to provide more definitive data. Several small series have been reported using GMCSF injected peri-lesionally in venous stasis ulcers. The largest trial for this growth factor was prematurely stopped due to systemic complications.
Combinations or sequences of growth factors are being tried. A recent report of sequential use of GMCSF and bFGF intent to treat growth factors showed that growth factor treated patients did better during the 35-day trial and were significantly more commonly healed at one year follow-up.
Reviewing the results of these various clinical trials suggest that growth factors may yet prove to be useful clinical adjuncts for wound healing.

GROWTH FACTORS, INCLUDING PDGF, IN WOUND HEALING
David Steed, Director, Wound Healing / Limb Preservation Clinic, Pittsburgh, USA

Growth factors are polypeptides which control cellular growth, differentiation and proliferation, and protein production. Their actions affect the inflammatory, proliferative, and maturation phases of wound healing. Growth factors stimulate cellular proliferation, chemotaxis, angiogenesis, and enzyme and matrix protein production.
PDGF is produced by platelets, 1fibroblasts, vascular endothelial cells, macrophages, and smooth muscle cells. It is a potent chemotactic factor and mitogen for inflammatory cells, fibroblasts, and smooth muscle cells. Wounds treated with PDGF have a marked increase in the number of inflammatory cells, leading to an increase in granulation tissue formation.
PDGF has been tested in the treatment of diabetic neurotrophic ulcers in a randomized prospective double blind trial. The healing rate was nearly doubled in the group treated with rhPDGF-BB, 48% versus 25% in the control group (P<0.01). This was the first demonstration of a benefit from a topically applied growth factor in the treatment of wounds. PDGF is now approved for this indication and is known as REGRANEX.
It appears that PDGF is most effective when applied to wounds which have been widely debrided initially and frequently during the course of treatment. Most wounds heal with good care. If they do not, there are new treatments available including growth factors, which modify the wound environment.

BIOTECHNOLOGY AND HEALING

GENE THERAPY OF WOUNDS AND ITS APPLICATION IN FUNCTIONAL GENOMICS
Jeff Davidson, Professor of Pathology, Vanderbilt University School of Medicine, Nashville, USA

It has become well established in the last decade that the wound is amenable to manipulation by the local administration of expression vectors thatencode a variety of growth factors and other gene products. Transient transfection can be accomplished in vivo with physical techniques such as the gene gun or liposomes, or by biological delivery with virus. Significant effects are obtained in some cases with single doses of naked DNA, suggesting that short-term expression initiates a response cascade. Viral delivery methods can lead to long-term expression in a variety of cell types. Therapeutic genes are not confined to secreted factors acting in trans: growth factor receptors, transcription factors, and components of intracellular signalling also show therapeutic potential. Ex vivo transfection of skin equivalents is an alternative mechanism for local delivery of secreted factors. Transfection of wounds is also a practical and efficient method for establishing wound healing activities as part of a gene discovery program that is designed to identify transcripts associated with early stages of injury and repair.

REPAIR IN THE LIVER

STEM CELLS IN THE LIVER
Heather Crosby, Liver Research Labs, Queen Elizabeth Hospital, Birmingham, UK

The liver, essentially a quiescent organ, is able to regenerate following resection or injury. If, however, hepatocytes are unable to proliferate due to disease or carcinogen exposure, a bipotential liver stem cell population is activated giving rise to hepatocyte and biliary epithelial cell lineages. The origin of these stem cells is not fully elucidated but recent studies in both rodent and humans suggests that cells of haematopoietic lineage may have the potential to differentiate into hepatic epithelium.
We have investigated this in human liver using the haematopoietic markers c-kit and CD34 to isolate sub-popu-lations of cells that differentiate into biliary epithelial and endothelial cells when grown in epithelial growth media. C-kit positive cells were identified from a range of normal and diseased tissue but attachment and subsequent growth of colonies varied.
C-kit cells were identified from donor tissue across an age range of 18 months to 51 years with an inverse correlation between the age and the ability of the cells to attach and proliferate. By contrast, mature biliary epithelial cells could be successfully grown from all ages of normal tissue.
This suggests that stem cells expressing haematopoietic markers may have the potential to regenerate liver epithelium and may provide an alternative source of hepatic cells for transplantation.

INFLAMMATORY CELL SIGNALLING, ROLES OF ANNEXINS
Francoise Russo-Marie, INSERM U332, Institut Cochin de Genetique, Paris, France

Annexin A1 (ANX A1) is the first member of a large and highly conserved family of proteins which are able to interact with membranes, particularly acidic phospholipids using calcium bridges. Recently, the full-length structure of ANX A1 has been uncovered by crystallization. Although ANX-A1 has the same characteristics of all other annexins, it has a remarkable feature. Its N-terminal domain forms, in the absence of calcium, a coil with two alpha helices embedded in the third domain of the protein core, whereas, in the presence of calcium, this N-terminus is cleaved, giving raise to a core protein and a 25 amino acids peptide.
The in vivo functions of ANX A1 are not fully understood but it is known that this protein family plays important roles in inflammation, membrane aggregation processes and in cellular traffic. ANX-A1 is also involved in proliferation, differentiation and apoptotic processes in epithelial and liver cells.
We will present evidence that ANX-A1, as a Janus-face protein, possesses two different functions:
o when extra-cellular, it becomes an acute phase protein, under the control of IL-6 and glucocortico-steroids, involved in the resolution of inflammation,
o when intra-cellular, and depending on the cell type,
it is involved in inflammatory signalling or in cell proliferation-differentiation.
The differential role of the N-terminus and of the core will be discussed.

BACTERIA AND WOUNDS

CHRONIC WOUND HEALING MICROBIOLOGY: AN OVERVIEW
Martin C. Robson, Professor Emeritus of Surgery, University of South Florida, USA

Health is not a germ-free state. Rather it is an equilibrium between the factors of host defense and a myriad of bacterial organisms. Each of the cellular processes of the wound healing scheme as well as the humoral mediators or messengers have been shown to be affected by bacteria. The effects of bacteria resemble the ying-yang effect demonstrated for pharmaceutical agents. A little bacteria appear to accentuate most of the reparative processes, while large amounts of bacteria inhibit the normal healing trajectory. Numerous studies have demonstrated that >105 bacteria/gram of tissue not only impair the specific processes, but also impair clinical healing. Studies have shown that closure of wounds by direct edge approximation, application of a skin graft, or rotation of a pedicle flap are not successful in the presence of an excessive bacterial burden.
Recent data demonstrate that humoral mediators such as growth factors are also impaired by >105 bacteria/gram of tissue. Proteases and other mmps produced by the bacteria and the interaction of bacteria and tissue cleave the molecules of both the growth factor proteins and their receptors.
This quantitative effect of bacteria appears to hold for most bacterial species. On beta hemolytic streptococci appear to inhibit healing processes at lesser levels. Because of the demonstrated effects, most clinical trials of new therapeutics drugs and/or devices try to control the bacterial burden in the wound prior to entry into the trial. The problem is how to decrease the bacterial burden and safely place the wound into bacterial balance. Wounds cannot heal or be closed with excessive bacteria. Lowering the bacteria can temporarily injure the wound. A balance must be struck between these apparent contradictions to move wounds toward an ideal healing trajectory.

MOLECULAR CHARACTERISATION OF COMPLEX BACTERIAL COMMUNITIES, INCLUDING THE UNCULTURABLE COMPONENT
William Wade, Guy's Hospital, London, UK

Culture has been the principal method of bacterial detection for the past 150 years. However, from the beginning it has been recognised that it is impossible to recreate in the laboratory the growth conditions required by all bacteria. In fact, on the earth as a whole, we are only able to culture around 2% of bacteria. A substantial component of the bacterial communities which cause disease in humans is also unculturable and it is therefore likely that as yet uncharacterised organisms are involved in disease processes. Molecular methods have now been developed to characterise bacterial communities in their entirety without the biases of culture. DNA is extracted direct from biomass and housekeeping genes, typically those encoding ribosomal RNA are amplified by PCR. The resultant mixed products are cloned and sequenced and the sequences compared to those held in nucleotide databases. In this way, bacteria in clinical samples can be identified without the need for culture. The microflora associated with a variety of oral infections have now been characterised. Numerous novel bacterial lineages have been detected and include some phyla normally associated with extreme environments. Molecular characterisation will enables the complete description of the normal and disease-associated microflora of man.