EUROPEAN  TISSUE  REPAIR  SOCIETY

INCREASED EXPRESSION OF IFN-g and TNF-a IN RADIATION- IMPAIRED WOUND HEALING
M. Schäffer¹, B. Proksch¹, W. Badach² and H.D. Becker¹ , 1 Department of Surgery, 2 Department of Radiotherapy, University of Tübingen, Germany

Background: Radiation impairs wound healing, the mechanisms leading to diminished repair, however, are poorly understood. Radiation decreases repair-promoting growth factors in the wound, such as PDGF, the effect on counter-regulatory inflammatory cytokines in unknown. We therefore hypothesised that irradiation may affect INF-g and TNF-a wounds.
Methods: Groups of 10 SD rata were electron-irradiated at the left side of their back with 12 Gy or sham-irradiated. Five days later animals underwent a dorsal skin incision in the irradiated skin area and PVA sponges were inserted subcutaneously. Animals were sacrificed ten days later to determine wound breaking strength (WBS) and sponge hydroxyproline content, an index of reparative collagen deposition. Cytokines were measured in wound fluid by ELISA.

N=10, mean ± SEM, *p<0.05 vs Control, #p<0.01 vs Control, ANOVA

Results: Radiation inhibited wound healing. Gain of body weight, total leukocyte count, differential leucocyte count and CD4/CD8 ratio were similar among the groups, suggesting that electron irradiation had little systems effect. Impaired healing was reflected in increased levels of IFN-g and TNF-a in wound fluid. Previously, we have demonstrated both diminished growth factor and nitric oxide expression following radiation.
Conclusion: Radiation does not uniformly decrease mediator expression due to non-specific DNA affection. In the wound, radiation causes an imbalance of decreased repair-promoting mediators and increased counter-regulatory factors, such as IFN- and TNF-, leading to diminished repair.

THE INFLUENCE OF MICRO-ENVIRONMENT AND CELL PHENOTYPE ON VENOUS ULCER HEALING
M.C. Stacey, M. Woosey, H. Wallace, K. Subramaniam and S. Yeoh, University Department of Surgery, Fremantle Hospital, Fremantle, Western Australia, 6160

The aim of this study was to compare the rates of proliferation of fibroblasts obtained from chronic venous leg ulcers with fibroblasts from normal skin and foreskin under normal culture conditions and in the presence of acute or chronic wound fluid.
Fibroblasts were obtained from three chronic venous leg ulcers, leg skin of three age-matched patients and foreskin from three infants. Acute wound fluid (AWF) was pooled from four mastectomy patients as was the chronic wound fluid (CWF) from four venous leg ulcer patients. Early passage (P3-4) fibroblasts from all cell lines were then incubated in either DMEM or DMEM with 10% fetal calf serum (FCS) in the presence of increasing concentrations (up to 25%) of AWF or CWF. At 48 hours cell numbers were determined using a crystal violet assay. Cell function was also assessed by incubating different fibro-blasts with acute and chronic wound fluid and measuring MMP-1, MMP-3 and TMP-1 in the supernatant.
Foreskin fibroblasts proliferated at a greater rate than either ulcer or normal age matched cells (ANOVA, P< 0.05). Normal age matched cells proliferated at a greater rate than ulcer cells in DMEM/FCS but not in DMEM alone. AWF and CWF at low concentrations (< 3.125%) stimulated similar proliferation in all cell lines. At higher concentrations <3.125% of AWF, proliferation was significantly less for ulcer cells (p<0.05). In contrast to AWF CWF at higher concentrations did not increase proliferation rates in all three cell types. Chronic wound fluid induced greater production of MMP-1 and MMP-3 from all cell types, and venous ulcer fibroblasts produced more MMP-1 and MMP-3 than other cell types.
Impaired wound healing in chronic venous leg ulcers is in part due to the decreased ability of local fibroblasts to proliferate and to the reduced ability of wound fluid to stimulate proliferation.

IN VITRO AND IN VIVO EVALUATION OF A SYNTHETIC COPOLYMER
A.J. van den Bogaerdt1,2, M. van Galen1, E.N. Lamme2 and E. Middelkoop1, 1: Research Department of the Dutch Burns Foundation, Beverwijk and 2: Isotis BV, Bilthoven, The Netherlands

Aim of this study: To improve the quality of regenerated tissue by using a biocompatible synthetic copolymer as a dermal substitute in full thickness wounds.
Materials and Methods: Dynamic and static seeding techniques were used to seed to copolymer matrices with human and porcine fibroblasts. Seeding efficiency and seeding density were evaluated as well as proliferation and extracellular matrix production. Seeded copolymer matrices were also transplanted on pigs in a full thickness wound model. Wound contraction was analysed by planimetry and wound biopsies were taken to analyse wound healing in a time frame of four weeks.
Results: Dynamic and static seeding reached comparable efficiencies (range 45-70%). The typical proliferation rate of human and porcine fibroblasts measured within the matrices was 4-6 times the initial seeding density of 100,000-300,000 fibroblasts/cm2 within 14 days. Fibro-blasts proliferated in time to a maximum level, which was independent on the initial seeding density. Extracellular matrix, formed by porcine fibroblasts, was observed in the matrix 21 days after seeding. The synthetic copolymer implanted as a 'living' dermal template was not able to prevent wound contraction and improve scar quality in porcine wounds, even when the matrices were pre-cultured during 21 days to induce EMC formation before implantation. Mean contraction of wounds treated with the dermal equivalent was 50-55% after four weeks, cell seeding resulted in a reduction of contraction of 10-15%. Within four weeks, large parts of the copolymer were extruded from the wound.
Conclusion: Although the copolymer has excellent qualities for cell culture in vitro, in vivo interactions between the material and keratinocytes negatively influenced wound healing.

ARTIFICIAL SKIN SUBSTITUTES SEEDED WITH FIBRIN GLUE CULTURED KERATINOCYTE SUSPENSIONS
Lachlan J. Currie and Robin Martin, Blond McIndoe Centre, East Grinstead, UK

Fibrin glue has a haemostatic effect after debridement, it improves split skin graft 'take' on difficult sites and it may have a protective role against bacterial graft destruction. Fibrin glue has also been used successfully as a delivery system for culture autologous keratinocytes (CAK) in suspension. CAK have been shown to produce a surface epithelium when seeded as a suspension into Integra_ artificial skin (Ethicon). In this study we investigated whether fibrin glue could be used to seed the underside of Integra_ with CAK in an effort to combine the beneficial effects of cell seeding with fibrin adhesion.
Five pigs (30 wounds) were used for this study. Ten wounds were grafted with Integra_ impregnated with CAK suspended in 1ml of growth medium, ten wounds grafted with Integra_ impregnated with CAK suspended in 0.5ml of fibrinogen/aprotinin component of Tisseel fibrin glue (Baxter) mixed with 0.5ml of growth medium. In this group the thrombin component of the fibrin glue kit was applied to the wound bed immediately prior to grafting. The remaining wounds were grafted with Integra_ either with or without fibrin. Epithelial cover was assessed in whole wound biopsies at 1, 2 and 3 weeks after grafting by histology and immunohistochemistry.
Fibrin glue provided rapid adhesion of the Integra_ artificial skin to the wound bed and there was a subjective reduction in haematoma formation. There was no detrimental effect on the Integra_ take rate, and there was an equivalent % of epithelial cover at a given time point.

DIABETES

MATRIX METALLOPROTEINASE (MMP) AND TISSUE INHIBITOR OF METALLOPROTEINASE (TIMP) EXPRESSION IN DIABETIC AND VENOUS ULCERS
Alan A. Rogers1, Edward B. Jude2, Samson O. Oyibo2 and Andrew J.M. Boulton2, 1: Wound Healing Research Institute, ConvaTec Global Development Centre, Deeside, UK; 2: Dept of Medicine and Diabetes, Man-chester Royal Infirmary, Oxford Road, Manchester, UK

Background and Aims: In venous leg ulcer (VLU) a defective balance between proteolytic enzymes and proteinase inhibitor expression, together with the underlying pathology results in delayed healing. In vivo, MMP's are regulated by TIMPs their natural inhibitors. Little is known of the pattern of expression of MMPs and TIMPs in diabetic foot ulcers (DFU).
Patients and Methods: Biopsies were taken from lower limb skin of non-diabetic subjects and the dorsum of the feet of diabetic neuropathic subjects and from the edge of neuropathic DFU and VLU. Frozen tissue sections were stained for MMPs-1, -2, -8, -9, TIMPs-1 and 2 in normal and diabetic skin ( NS and DS, respectively ) DFU and VLU.
Results: All MMPs and TIMPs were detected in the base of all ulcers examined, with MMP-8 and TIMP-2 showing intense staining. MMP-9 expression was particularly strong within VLU. MMPs-1 and 8 were expressed in NS and DS, particularly in epidermal cells. An increase in MMP-8 staining was seen in the dermis adjacent to DFU and VLU. There also appeared to be increased basal epidermal cell staining for MMP-1 and TIMP-2 at the ulcer margins. TIMP-2 was located weakly in the dermis and along regions of the epidermal-dermal basement membrane of NS and DS. Within the ulcer base of DFU and VLU, MMP and TIMP expression was similar. However, at the edge of VLU there was enhanced staining of MMPs-2,-8,-9 and TIMP-1 in the dermis under the epidermal leading edge.
Conclusions: Both MMP and TIMP expression appears elevated in chronic wounds and surrounding skin. Abnormal and excessive expression of proteinases and their inhibitors may play a role in the non-healing nature of chronic wounds by upsetting the proteinase-inhibitor balance necessary for healing to occur.