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FIBROBLAST SUBPOPULATIONS IN INTRA-ORAL WOUND HEALING
H.E. van Beurden, J.W. Von den Hoff and R. Torensma, UMC Nijmegen, The Netherlands

The objective of this study was to characterise wound fibroblasts from sequential time points during intra oral wound healing in the rat.
Wounds were made with 3mm biopsy punches in the mucoperiosteum of the palate of 35-day-old Wistar rats. Wound biopsies were taken at 3, 5, 8, 15, 30, 60, 90 and 120 days post-wounding. Fibroblast populations were isolated from each biopsy and cultured under standard conditions with the same number of passages. The expression of the integrins a1 a6 b and the intermediate filaments vi-mentin and a-smooth-muscle actin were analysed by flow-cytometry. In addition, cryosections of the wound areas were made at the same time points. These sections were immunohistochemically stained for the same antigens.
The expression of a1 and b was increased in fibroblast cultures from biopsies taken at 5 days post wounding and a-smooth-muscle actin and vimentin were highly increased between 8 and 15 days post wounding. The immunohistochemical expression of a-smooth-muscle actin in cryo-sections of the wound area corresponded to the flowcyto-metrical data. These results indicate the existence of multiple subpopulations of fibroblasts with a specialised function during different time points in wound healing. These specialised phenotypes of fibroblast populations appeared to be conserved during in vitro culture.

THE PREVENTION OF ACUTE FASCIAL WOUND FAILURE
Michael Franz, M. Ann Kuhn, Xue Wang and Martin Robson, University of Michigan, Ann Arbor, MI, USA

Abdominal wall wound healing failure remains a significant surgical problem. Prospective series report an 11% incidence of primary fascial wound healing failure leading to incisional hernia formation. The incidence of hernia re-currence following repair is even greater (24-58%). This study was designed to characterise the pathophysiology of incisional hernias and to test whether accelerated fascial repair will reduce the incidence of this common surgical complication.
A rat model of acute ventral abdominal wall myofascial incisional healing was used. In Group 1 (n=20), the fascial closure was securely closed with non-absorbable suture. In Groups 2 and 3, the fascial layer was primed with vehicle or TGF-b2 prior to incision. Animals were sacrificed on PODs 0, 1, 7, 14, 21 and 28 and the incidence of herniation and wound breaking strengths were measured. Fascial fibroblast kinetic and synthetic activity was determined by FPCL, immunostainings and RT-PCR for collagens I and III.
Unimpaired fascial breaking strength recovers earlier than dermis. No incisional hernias developed in Group 1. In Group 2, there was an 88% incidence of incisional hernia formation. No hernias were observed in Group 3 (TGF-b2 treated). Hernia fibroblasts achieved 74% less collagen matrix contraction than normal fascial fibroblasts. Uncomplicated fascial healing was associated with a more organised provisional matrix and increased collagen I and III immunostaining. Collagen gene expression was the same at 28 days.
Acute fascial wound failure leading to incisional hernia formation is an early biomechanical event. A deficiency exists in hernia fibroblast kinetic and synthetic activity. The mechanism of incisional hernia formation may involve the loss of normal mechano-transduction signals for repair fibroblast activation. Therapy of incisions with TGF-b2 can correct the defect.

INCREASE IN SUBCUTANEOUS OXYGEN WITH OXYGEN BREATHING DURING ACUTE SEVERE ISOVOLEMIC ANEMIA
H.W. Hopf, J. Feiner, M .Viele, J. Watson, R. HO, R. Weiskopf, T.K. Hunt and P. Toy, Departments of Anesthesia and Perioperative Care, Surgery, Laboratory Medicine, Physiology, University of California, San Francisco, CA USA (Supported by NIH NHBLI SCOR HL55476)

Introduction: Subcutaneous wound tissue oxygen tension (PsqO2) is maintained in healthy volunteers during acute isovolemic hemodilution (HD) to hemoblobin (Hb) 5 g/dL by an increase in subcutaneous blood flow, as evidenced by a significant increase in subcutaneous temperature (Tsq). In volunteers with normal Hb, PsqO2 increases from 65 to 100-130mm Hg when PaO2 increases from 100 to 300-400mm Hg. We hypothesized that the response to an increase in PaO2 would be greater at Hb 6 g/dL than at normal Hb because of the increase in subcutaneous blood flow.
Methods: Seven healthy volunteers underwent HD. Shed blood was replaced with 5% albumin and autologous plasma to maintain normovolemia. PsqO2 and Tsq were measured at baseline with subjects breathing 15 Lpm air, and at nadir Hb after 20 min breathing 15 Lpm O2 or air (randomised) via face mask. Red cells were reinfused overnight.

Results are Mean ±SD except PsqO2 is median (range).
*p<0.02 vs.room air.

Conclusions: PsqO2 increased significantly during oxygen breathing. The increase was greater than that seen previously in volunteers with normal Hb concentration. Administration of supplemental oxygen to patients with anemia may have a greater impact on wound healing than in patients with normal Hb concentration.

PROTEINASE AND GROWTH FACTOR PROFILES IN ACUTE COLORECTAL WOUND FLUID
E.A. Baker and D.J. Leaper, Professorial Unit of Surgery, University Hospital of North Tees, Stockton on Tees, UK

Background: Wound healing is a complex process involving complex interactions between different cell types, extracellular matrix components and biological factors. The aim of the study was to determine the daily profiles of matrix metalloproteinases (MMP-1, -2, -3, -9), tissue inhibitors of metalloproteinases (TIMP-1, -2), cytokines (IL-6, IL-1b, TNF-_) and growth factors (EGF, PDGF, VEGF, TGF-b1, bFGF) in acute wound fluid collected post-operatively from the surgical drain of colorectal patients (n=52). The levels of these factors were correlated with clinical features (Dukes' stage, stoma, post-operative complications).
Methods: Samples were analysed by gelatin zymography (MMP-2, -9), ELISA (MMPs/TIMPs (ng/ml), growth factors (pg/ml)) and quenched fluorescent substrate hydrolysis (total MMP activity).
Results: Differential levels of these factors were observed on each day, e.g., day 1; MMP-3: 58, median (2-447, range); TIMP-1: 5729(519-14860); EGF: 87(3027-19444); TGF-b1: 6937(3027-19444). A significant negative correlation was observed with MMP-3, TIMP-2, TIMP-1, total MMP activity, IL-6, EGF, PDGF, bFGF and TGF-b and the post operative day (P<0.05 Spearman's correlation) e.g., TIMP-2: day 1, 168(1-758), day 3, 121 (6-596), day 7, 46(0-101). On days 1-6, TNF- and IL-1b levels were significantly greater in patients with stomas (P<0.05). Correlations are emerging between the levels of several factors and post-operative complications.
Discussion: Colorectal wound fluid demonstrated differential profiles of proteinases, inhibitors and growth factors over time and with clinical outcomes. The balance in the levels of these factors may affect the stages/rate of wound healing in vivo.

MECHANISMS IN THE PATHOPHYSIOLOGY OF PROGRESSIVE BURN INJURY: THE PROTEASE/ANTI-PROTEASE IMBALANCE
Paul E. Banwell, T.S.T. Adams, S.E. Herrick and D.A. McGrouther, Stoke Mandeville Burns & Reconstructive Surgery Research Trust, UK

Introduction: Although the concept of progressive dermal damage following thermal trauma is well established, the exact mechanism for tissue destruction is unknown. Recently, neutrophil degranulation products (proteases) have been implicated in models of tissue destruction, although their relevance in the burn wound is unknown. We therefore examined the expression of neutrophil proteases in human partial thickness Burns.
Patients & Methods: A prospective, longitudinal human study was performed on eighteen patients (n=18) with partial thickness burns. All burns were clinically assessed on admission and at 48 hours when tissue biopsies were taken> Progressive injury was confirmed histologically and neutrophil profiles recorded using targeted antibodies (CD15). Neutrophil proteases (neutrophil elastase, cathepsin G, MMP-8, MMP-9) and their inhibitors (_1-PI, TIMPs) were analysed using biochemical assays and immunohistochemistry.
Results: Histology confirmed vascular injury and matrix damage with associated burn depth progression. Depth of injury correlated with the total number of neutrophils (CD15) (p<0.0001, Mann-Whitney) at 48 hours. Assays confirmed an abnormal protease/antiprotease profile with high levels of free protease (p<0.0001, ANOVA).
Conclusion: This study demonstrates that progressive dermal damage is associated with neutrophil extravasation and increased expression of neutrophil proteases in the first 48 hours following cutaneous burn injury. Restoration of the protease/antiprotease balance may represent a novel method for burn wound treatment.

NEUTROPHIL-DERIVED ELASTASE EXPRESSION IN HUMAN PARTIAL THICKNESS DERMAL INJURY DURING TREATMENT WITH TOPICAL NEGATIVE PRESSURE
T.S.T. Adams, P.E. Banwell, S. Herrick and D.A. McGrouther, Stoke Mandeville Burns Reconstructive Surgery Trust, Aylesbury, UK

Background: Neutrophil Elastase (NE) is thought to contribute to the proteolytic activity noted in difficult-to-heal wounds, including burns. Using a novel Topical Negative Pressure (TNP) device, we investigated the temporal NE profile in human split skin graft donor sites and burn wounds during the first 48 hours of injury.
Methods: A prospective longitudinal study was conducted in patients admitted to the Oxford Regional Burns Unit. Wound fluid was collected in patients (n=5) incorporating an irrigation system> Between 1^ and 20 samples were collected per patient. Wound fluid was processed in a standard fashion and elastase activity quantified using a synthetic elastase substrate (methoxysuccinyl-ala-ala-pro-p-nitroanilide, Calbiochem) colorimetric degradation assay, against standard human elastase concentrations (expressed as ng/100mcg protein).
Results: In human donor site wounds, peak elastase concentrations (4.3.4ng ± 4.97, mean ± SEM) occurred between 12-18 hours post wounding and demonstrated a mean percentage decline of 63% over 48 hours. In human partial thickness burns, elastase increased over the same period (mean percentage increase of 92%) with peak concentrations (45.6ng ± 1.6, mean ± SEM) from 28 to 40 hours.
Conclusions: Using an irrigated TNP device, we have demonstrated differential temporal profiles of NE in human donor site wounds compared to burns. These results suggest that burn wound fluid exhibits proportionally prolonged protease activity that might account for differences in observed healing rates.