WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001138 Meeting Schedule and Abstracts

1. 3.

PLATELET-DERIVED GROWTH FACTOR-B CHAIN DNA PLASMIDADENOVIRAL OVEREXPRESSION OF SOLUBLE GROWTHFACTORS AND THEIR EFFECTS IN IMPAIRED WOUND HEALING. AUGMENTS THE SURVIVAL OF ISCHEMIC MYOCUTANEOUS FLAPS:

A ROLE FOR GENE THERAPY IN PLASTIC SURGERY.J Karmacharya, A Gordon, F Lim, B Martin, B Edelman, A Radu, CGruss, K Satyamoorthy, M Herlyn, and TM Crombleholme. J. Hijjawi, I. Kim, J. Mogford, L. Chandler, J. Ma, D. Gu, G. Pierce, T.

Mustoe.Children’s Institute for Surgical Science at the Children’s Hospital ofPhiladelphia, The Wistar Institute, The Univ. of Pennsylvania, Northwestern University Medical School, Div. of Plastic Surgery,

Chicago, IL and Selective Genetics Inc., San Diego, CA.Philadelphia, PA.

This study aims to evaluate the efficacy of platelet-derived growthNormal wound healing is a complex process involving highly regulatedcascade of events requiring interactions between many cell types, factor B chain (PDGF-B) plasmid DNA embedded in a collagen matrix

(1% fibrillar collagen, 1% gelatin), in augmenting the survival of ischemicmatrix components, and soluble factors. Deficiencies in any of thesecomponents result in impaired wound healing. The purpose of these transverse rectus abdominis myocutaneous (TRAM) flaps in rats.experiments are to screen soluble growth factors in an impaired woundhealing environment and identify specific factors that markedly PDGF-B plasmid DNA formulated in a collagen matrix (800 microliters

at 0, 1.5, 3.0 and 6.0 mg/ml) was injected into the skin paddles ofmodulate the wound healing response for further evaluation. Weprepared adenoviral vectors carrying the genes for 18 different soluble inferiorly-based TRAM flaps in female Sprague-Dawley rats (n � 28).

Seven days later, these TRAM flaps were elevated and sutured in situgrowth factors having known effects and others that have not beenpreviously characterized in wound healing. Seven days after direct with an underlying silicone sheet to prevent vascular ingrowth from

the abdominal wall. On post-operative day 7 (14 days after injectioninjection, 10 mm excisional wounds in diabetic C57Bl/KsJ-db/db mice(n � 100) were evaluated and histological scored for epithelialization, of PDGF-B plasmid DNA), a survival ratio was calculated based on

the percentage of the original flap remaining viable. Our study revealedangiogenesis, matrix deposition, and inflammation by 3 blindedobservers. Marked re-epithelialization was noted in defects treated with a dose-dependent effect of PDGF-B plasmid DNA on the survival of

the ischemic rat TRAM flap. Control animals treated with 0 mg/mlTGFb1, PDGF-A, PDGF-B, VEGF and Ang -1 treated defects.Angiogenesis was noted to be increased with, PDGF-A, PDGF-B, Ang (collagen matrix alone) demonstrated a survival ratio (SR) of 24 � 7%

(ave � SEM), while those treated with 1.5 mg/ml, 3.0 mg/ml and 6.01, MMP-9 (matrix metalloproteinase), but surprisingly large number ofblood vessels were increased in defects treated with serine protease mg/ml demonstrated survival ratios of 44 � 5%, 49 � 3% and 54 �

4%, respectively.uPA. Reduction in the inflammatory response was noted in defectstreated with cytokines Il-10, Il-8, pliotrophin, IGF, tPA, and HPV16-E6

These results clearly indicate that PDGF-B plasmid DNA delivered in(Human papiloma virus type E6). Matrix deposition was increased indefects treated with PDGF’s, TGF’s, Ang-1, and cell adhesion molecule a collagen matrix augments the survival of ischemic myocutaneous

flaps in a dose-dependent fashion. In previous reports, the PDGF-BBMMC18. Marked inflammatory response was noted in defects treatedwith adenoviral vectors carrying the reporter gene Lac Z. Adenoviral protein has been shown to improve the healing of chronic, ischemic

lower extremity wounds in diabetic patients and experimentally, tomediated overexpression of soluble growth factors in the diabeticmouse model of impaired wound healing is a useful screening system augment the survival of the mouse latissimus dorsi muscle flap. We

have demonstrated similar augmentation of myocutaneous flap survivalto characterize known effects and identify novel effects of new solublefactors in wound healing. These results identify several promising by providing localized gene expression in a collagen matrix. The

collagen matrix localizes plasmid DNA within the flap and provides aadenoviral constructs for future studies of gene transfer in woundhealing. scaffold for cellular infiltration, thereby enabling target cell

transfection. Furthermore, matrix-enabled gene therapy is ideallysuited for plastic surgery applications where only transient therapeutic

2. transgene expression is required, i.e. during the limited time in whichADENOVIRAL MEDIATED OVEREXPRESSION OF INSULIN-LIKE tissue is under ischemic insult in myocutaneous flaps and in chronicGROWTH FACTOR-1 (IGF-1) PROMOTES DOSE-DEPENDENT RE- wounds.EPITHELIALIZATION IN ISCHEMIC AND NON-ISCHEMIC WOUNDS.

B Edelman, M DelPino, J Karmacharya, A Gordon, F Lim, B Martin, A 4.Radu, R Kirschner, K Satymoorthy, M Herlyn, and TM Crombleholme. DNA-GROWTH FACTOR TRANSFECTION TO ENHANCEChildren’s Institute for Surgical Science at the Children’s Hospital of CUTANEOUS WOUND HEALING.Philadelphia, The Wistar Institute, The Univ. of Pennsylvania,Philadelphia, PA. CK Byrnes, FH Khan, PH Nass, C Hatoum, MD Duncan & JW Harmon.

Department of Surgery, Johns Hopkins Medical Institutions, Baltimore,We have previously demonstrated the ability of adenoviral-mediated MD.IGF-1 (Ad-IGF-1) overexpression to enhance re-epithelialization inischemic wounds. The purpose of this study was to determine if Our group and others have reported enhancement of cutaneous wound

healing following the transfection of tissue with plasmid vectorsoverexpression of IGF-1 enhances epithelialization and granulation ina dose-dependent manner. Ischemic ears were created by arterial expressing the DNA for growth factors (Sun et. al. J Invest Dermatol

108: 313-8, 1997). In these prior DNA transfection experiments, growthligation in 10 to 12 week old New Zealand white rabbits (n � 8). After24 hours, four 6 mm excisional full-thickness skin wounds, including factor treated animals were usually compared to animals treated with

control plasmid vector without the specific growth factor gene.perichondrium but preserving the cartilage, were created on ischemicand non-ischemic ears. Wounds were treated by direct injection of Ad- However to show that there is true enhancement in wound healing,

the pertinent control is an untreated wound. We performed experimentsIGF in escalating logarithmic doses (10isto4 to10-10 PFU) and harvestedat seven days for morphometric analysis of histology. Histological to see if the high DNA plasmid load and repeated needle penetrations

of the wound required to achieve consistent transfection wereexamination documented enhanced epithelialization and granulationwith increasing doses of Ad-IGF-1. The epithelial gap in wounds treated detrimental to wound healing.with Ad IGF-1 at PFU’s of 104. 106, 108 , 10-10 PFU was 3.5mm, 2.3mm,0mm, 0mm respectively. The epithelial gap was 2.9 mm (control) and Diabetic C57 mice, which exhibit delayed wound healing, received full

thickness 5 mm excisional wounds, and were assigned to one of the6. 5 mm in defects treated with Adlac Z at 108 PFU in ischemic ears.The granulation tissue formation in mm. Sq for controls are 53.7mm2, following groups, no treatment, PBS injections, and plasmid vector

injection with and without the KGF gene. Intradermal injections ofand 15.8 mm2 defects treated with Adlac Z at 108 PFU. The granulationin wounds treated with Ad IGF-1 at PFU’s of 104. 106, 108 , 10-10 PFU 100ug of plasmid adjacent to the wounds were given at days 1-5, 7 and

11 post wounding. Transcription was confirmed using RT-PCR for KGFwas in 63.8 mm2, 117 mm2, 40.1 mm2, and 189 mm2 respectively. Inconclusion, adenoviral mediated gene transfers of Ad-IGF-1 influences mRNA. At day 9 the unepithelialized wound area (pixels) was measured,

and on day 17 the wounds were excised and burst strength (Newtons)re-epithelialization and granulation in a dose-dependent manner andmay prove useful in wound healing. measured.

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 139At day 9, wound closure was more advanced in the KGF-1 treated ends of eukaryotic chromosomes. By preventing telomere shortening

that signals replicative senescence, exogenous expression of hTERT,animals (wound area � 2719 � 304) than in the animals treated withcontrol plasmid (wound area � 5506 � 501 p < 0.003). But a detrimental the catalytic component of telomerase, can extend the replicative

lifespan of human somatic cells (including fibroblasts, endothelial cellseffect of the DNA plasmid injection was evident from a comparisonof the DNA control group (wound area � 5506 � 501) versus the non- and keratinocytes) while maintaining the phenotype and functions

characteristic of younger cells. To assess whether hTERT can rescueinjected group (wound area � 2509 � 457 p < 0.001). The effect wasa consequence of both the DNA injection and the repeated needle the defect in wound healing associated with skin aging or chronic skin

diseases, we have established an in vivo wound-healing model usingsticks, as evidenced by the PBS group results (3647 � /-537) fallingintermediately between the values for the no-injection and the plasmid SCID-Hu xenografts. We have grafted over 100 SCID mice with human

full thickness skin from over 30 donors (newborn to 92 yr. old).control groups. A similar effect was seen with wound burst strengths:plasmid control 1.21 � 0.077, KGF-1 plasmid 1.79 � 0.13 (KGF versus Immunohistochemical assays for markers of human and murine skin

(human involucrin, collagen I, collagen IV, HLA-A, B, C and murineplasmid control p < 0.016), non-injected 1.43 � 0.16 and PBS 1.51 �0.09. H2kd) were used to assess the structure and composition of the

xenografts. The xenografts were wounded by creating a biopsy woundThe challenge for developing an effective system for the enhancement and the rate of wound healing monitored for xenografts from young

and old donors. To deliver hTERT to the xenografts, several modes ofof wound healing lies in improving transfection efficiency. In this wayit will be possible to reduce the negative effect of multiple injections gene delivery to the transplanted xenografts are being evaluated: gene

gun and direct injection for hTERT plasmid DNA; direct injection orand plasmid load.topical application for adenoviruses containing hTERT. Telomeraseexpression by established TRAP assays has been detected with both5.

topical and injection methods. We will present results on the optimalCELL THERAPY FOR WOUND HEALING- STUDIES IN SKIN ANDroute of gene delivery and the effect of hTERT expression on the rateUPPER AIRWAY.of wound healing.

PA Hebda, S Nishimoto, FR Rosas, JE Dohar. Dept of Ped Otolarynogol,Children’s Hospital of Pittsburgh and Depts of Otolaryngol and Cell

7.Biol & Mol Physiol, Univ of Pittsburgh School of Med., Pittsburgh, PA. INHIBITION OF PROLYL 4-HYDROXYLASE REDUCES SCAR

ELEVATION IN A RABBIT MODEL OF HYPERTROPHIC SCARRING.Cell therapy and bioengineering hold great promise as a therapeuticapproach to treat various pathologic or trauma-induced states. One Injoong Kim, Jon E. Mogford, Claudia Witschi, Mehdi Nafissi, Thomas

A. Mustoe. Northwestern University, Chicago, IL.possible application is the transplantation of cells into wounded tissueto help regulate tissue repair. Our goal is to achieve less scarring and

Hypertrophic scars result from excessive collagen deposition at sitesmore regenerative healing through the selection of the best donor cellsand the best gene expression. There is strong support for the use of of cutaneous wounds causing functional and cosmetic problems.

Regulation of collagen synthesis/deposition is a direct approach to thefetal fibroblasts because of minimal host response to fibroblasts ingeneral, and because they are the key responding cell in scarless fetal control of scar tissue formation. We tested a prolyl 4-hydroxylase

inhibitor FG-1648 (0.5% Carbopol 971 PNF gel, pH 6.5) for reducinghealing. The specific aim was to characterize the host response to, andthe beneficial outcome of, fetal fibroblast donor cells, utilizing rabbit hypertrophic scar formation in a rabbit ear hypertrophic scar model.

Prolyl hydroxylase is required for stable native collagen formationmodels of skin and airway mucosal wound healing, and to comparethem with postnatal fibroblast donor cells. Fibroblasts obtained from through the catalyis of hydroxylated prolyl residues. New Zealand

White rabbits were divided into two treatment groups (n � 3 pertissue biopsies, were grown in culture and stored frozen at low passagenumber (P1 or P2) until use. Cells were thawed, grown briefly in culture group): low dose group: 0.5% prolyl hydroxylase inhibitor; high dose

group: 1% prolyl hydroxylase inhibitor. Left ears were used forthen labeled with a vital fluorescent dye. The cells were suspended insaline and delivered by syringe to the wound site. For skin wounds, treatment and right ear for control. Four 7-mm dermal ulcer wounds

were made on each ear. Prolyl 4-hydroxylase inhibitor FG-1648 wasfull thickness scalpel incisions were treated then covered withtransparent film dressing. For airway wounds, partial thickness curette topically applied to the wound at the time of wounding and once daily

up to post-operative day 7 (POD7). Wounds were harvested at POD28wounds were treated then covered with transparent hyaluronic acid-based, biodegradable film dressing. Control wounds were untreated and scar hypertrophy quantified by measurement of the scar elevation

index (SEI). Data were analyzed by Student’s t-test. Treatment ofbut covered with the wound dressing. Wounds were excised and freshfrozen for microscopic evaluation. Tensile strength of the skin incisional wounds with 1% FG-1648 decreased SEI by 23% compared to control

wounds (p < 0.01). Wounds treated with 0.5% FG-1648 showed nowounds was also measured. In both types of wounds the labeled cellswere detected up to 3wks following injury. There were no signs of an difference in scar elevation compared to control wounds. Wounds

harvested at POD12 demonstrated no difference between test groupsimmune response to the allogenic cells. There was evidence that thetransplanted fibroblasts participated in the wound healing response, regarding granulation tissue coverage of wounds (n � 9) or epithelial

gap (n � 9). These results suggest that prolyl 4-hydroxylase inhibitorbased on increase in tensile strength of the skin wounds and on woundmaturity of skin and airway wounds. The results prove that allogenic FG-1648 may be a suitable agent for topical treatment for the prevention

of hypertrophic scar tissue.fibroblasts survive in an immunocompetent host and function bycontributing to the wound healing response. These experimentsestablish the basic feasibility for using allogenic cell therapy for skin 8.

and airway wound healing. (Supported by Children’s Hospital of PHARMACOLOGICAL ACTIONS OF RECOMBINANT HUMANPittsburgh.) RELAXIN (RHRLX) IN IMPAIRED WOUND HEALING II:

VASODILATION IN H-SHAPED ISCHEMIC WOUNDS IN RATS.6.

G. Arnold, L. Guzman, J. Cheng, E. Unemori, and X. Huang. ConneticsTELOMERASE THERAPY ON A WOUND CLOSURE MODEL IN SCID-Corp., Palo Alto, CA.Hu SKIN XENOGRAFTS.

Our previous work demonstrated that recombinant human relaxinEdwin Chang, Adrienne Bronstein, Angela Jacks, Calvin Harley andChoy-Pik Chiu. Geron Corporation, 230 Constitution Drive, Menlo Park, (rhRlx) stimulated healing of impaired wounds in two models of dermal

wound healing, the wound chamber model and the H-shaped doubleCA, USA, 94025.flap model. The purpose of this study was to investigate thepharmacological effect of rhRlx in preclinical ischemic wound healingReplicative senescence of cells (keratinocytes, fibroblasts and vascular

endothelial cells) is associated with growth arrest and altered gene and to study the possible mechanism of action. A standardized H-shaped ischemic flap wound was made on the back of Lewis rats.expression in culture. Given certain similarities in the phenotype of

old cells ex vivo and in vivo, it is believed that replicative senescence RhRlx was delivered by subcutaneous infusion with Alzet� pumps (AlzaCorporation, Palo Alto, CA) implanted at the time of surgery. Thecontributes to the aging phenotype of human skin as well as defects

in wound healing associated with chronic skin conditions. Telomerase incidence of surface necrosis was evaluated and the area of necrosiswas quantitated, using Simple PCI image analysis software, for Day 10is a reverse transcriptase that adds repeats of TTAGGG to the telomeric

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001140 Meeting Schedule and Abstracts

and Day 14. RhRlx treatment reduced both incidence and area of of 3 female and 3 castrated male red Duroc pigs weighing 10-15 Kg,using an electric dermatome and trephine or scalpel, respectively. Thesurface necrosis compared to control animals. Incidence of surface

necrosis on Day 14 was: vehicle 23/30 (77%), rhRlx 14/30 (47%), p < wounds were bandaged for 24 hours, and then left open to healthereafter. At 2, 4, 6, 8, and 10 weeks post-surgery for the females, and0.05. Day 14 results for area of surface necrosis were: vehicle 1.15 cm2,

rhRlx 0.56 cm2, p < 0.05. In a subsequent study the nitric oxide synthase out to 26 weeks in the males, 4 mm punch biopsies were taken fromthe partial and full thickness wounds, as well as normal skin, forcompetitive inhibitor L-NAME was administered via the drinking water,

in conjunction with systemic rhRlx treatment. The reduction in histological examination, as well as for DNA and RNA quantificationand subsequent RT-PCR analysis for relevant molecules.incidence of surface necrosis and surface necrotic area observed with

rhRlx treatment alone was inhibited by L-NAME. Area of surfaceBoth the partial and full thickness wounds heal relatively rapidly,necrosis on Day 14 was: vehicle 1.91 cm2, vehicle � L-NAME 2.76

cm2, rhRlx � L-NAME 2.40 cm2. The data from these studies suggest demonstrating an immediate increase in the amount of RNA and DNAextracted from the tissue samples, and completing woundthat the systemic administration of rhRlx benefits the ischemic wound

via vasodilation, and that this vasodilatory effect may be mediated, at reepithelialization within 28 days of the initial surgery. Histologicalanalysis demonstrated that the wound healing then continued post-least in part, through the nitric oxide pathway.reepithelialization. Staining with hematoxylin and eosin, and with Siriusred in picric acid revealed that by day 42 the dermis was markedly9.

thickened, and extensive disorganized depositions of collagen wereTGF BETA RECEPTOR TYPE I EXPRESSION VARIES WITH DEGREEpresent. Marked, pathologic wound contracture and severe hyperOF SCAR FORMATION.pigmentation was also grossly apparent in both the females and in the

MJ Lee, JE Mogford, AS Saulis, TA Mustoe. Northwestern Univ. Medical castrated males in both the partial and full thickness wounds, inSchool, Div. of Plastic Surgery, Chicago, IL. contrast to that previously observed in a Yorkshire pig model. As well,

in both the female and male red Durocs, the full thickness woundsDysregulation of transforming growth factor (TGF)-beta isoforms have demonstrated a marked skin defect (involution) at and after 70 daysbeen implicated in scar pathogenesis. These isoforms must act on a post-surgery, a phenomenon not apparent in the partial thicknessreceptor complex of type I and II receptors where the type I receptor wounds.propagates the signal to downstream targets. We compared thetemporal expression of TGF beta 1, beta 3, and type I and II receptor The present study indicates that the healing of excisional wounds inmRNA expression in scars reduced by silicone occlusion versus the red Duroc pig differs from that in Yorkshire pigs, a finding that ishypertrophic scar in the rabbit ear hypertrophic scar model. Four 7mm consistent with the use of the red Duroc pig as a model of hypertrophiculcers were created on each ear of young, female New Zealand rabbits scar formation.and harvested on day 7, 28 and 56. (n � 3 for each group). Anothergroup (n � 3) was treated with silicone occlusion and harvested at

11.day 56. The scar elevation index showed a statistically significantEARLY BIOMECHANICAL WOUND FAILURE: A MECHANISM FORreduction in the silicone treated versus control group at d56 postVENTRAL INCISIONAL HERNIA FORMATION.wounding. RNA was extracted from each sample. RT-PCR was

performed and fluorometric quantification of product was done.Michael G. Franz, M. Ann Kuhn, Keoni Nguyen, Xue Wang, Francis Ko,Results are quantified as a percent expression compared to theTerry E. Wright and Martin C. Robson. University of Michigan, Anncyclophyllin gene found to be unaltered in wound healing and scarArbor, MI and Institute for Tissue Regeneration, Repair andformation. TGF-beta 1expression was greatest at day 7 (140 � 8%Rehabilitation, Bay Pines VAMC, Bay Pines, FL.SEM) and decreased until day 56. TGF-beta 3 expression started low

at day 7 (17.0 � 2%), increased at day 28 (108 � 2%) and then decreased.200,000 remedial operations are performed annually in the US to repairTGF beta type I receptor had elevated levels of expression at day 7(142primary ventral incisional hernias. It is estimated that the true incidence� 6%), which minimally declined except in the silicone-treated scar.of this iatrogenic problem is closer to 400,000. Recurrence ratesTGF beta type II receptor expression was found to be greater thanfollowing repair remain 24-51%. Despite its importance, the mechanism500% of control at all days of harvest with no difference between theand natural history of ventral incisional hernia formation andsilicone treated and hypertrophic scar. No difference in expressionrecurrence is unknown. This study was designed to measure thewas found between hypertrophic and silicone treated scar for TGFbiological and mechanical events leading to ventral incisional herniabeta-1 (79.1 � 7% vs. 77.0 � 6%) or beta-3 (91.8 � 19% vs. 77 � 2%).formation in a new animal model.Type 1 receptor expression was significantly reduced (p < .001) in the

reduced scar (7.0 � 6%) compared to the hypertrophic scar (116 �Forty Sprague-Dawley rats underwent elevation of a ventral abdominal3%). The temporal expression of TGF beta 1 and beta 3 confirms otherwall skin flap. An isolated midline fascial incision was then placedstudies which demonstrate TGF beta 1 expression early in woundthrough the linea alba during celiotomy. In Group I (n � 20) fascialhealing while TGF beta 3 expression occurs in scar formation.incisions were immediately closed with a single 4-0 nylon suture. InDecreased scar production has a significantly decreased expression ofGroup 2 (n � 20) fascial incisions were closed with a single fine fast-type I receptor. These results are consistent with the mechanism thatabsorbing suture (5-0 plain catgut). Ten rats from each group werethe type I receptor may be the regulating step in activating downstreamkilled on PODs 7 and 28 and necropsy performed. Incisional herniastargets in scar formation.were defined as a 2mm or greater fascial wound defect. Fascial woundbiopsies were obtained at each timepoint for standard histological

10.staining and immuno-histochemical analysis using antibodies againstTHE RED DUROC PIG MODEL OF HYPERTROPHIC WOUNDrat collagens type I and III. Rat collagen I gene expression was measuredHEALING.on POD 28 by RT-PCR.

Corrie L. Gallant, J. Barry Wright, Merle E. Olson, David A. Hart.Department of Microbiology and Infectious Diseases, Faculty of No incisional hernias developed in Group I by 7 or 28 days. Incisional

hernias were evident by POD 7 in Group II (7/10 at 7 days and 8/10 atMedicine, University of Calgary, Alberta CANADA.28 days; P < 0.05). Intensity of collagen I and III staining was the samewhen comparing Group I to Group II and when comparing healed toThe process of hypertrophic scar formation has not yet been well

characterized in vivo. The red Duroc pig has been suggested by herniated abdominal walls within Group II. No difference in rat collagenI gene expression was detected between healed and herniatedSilverstein et al. and Matsumura et al. to be such a model which, unlike

loose skinned animals such as mice and rabbits, results in the formation abdominal walls.of hypertrophic scars following the creation of acute excisionalwounds. Our laboratory has now developed a partial thickness and Early mechanical abdominal wall wound failure alone leads to ventral

incisional hernia formation in this model. Collagen gene expressionfull thickness excisional model in the juvenile red Duroc pig.and synthesis appear to be normal. The known lag period in therecovery of fascial wound relative breaking strength likely contributesTen partial thickness (1.8 mm), 2 cm � 2 cm wounds, and ten full

thickness, 2 cm diameter wounds were created on the dorsal surface to the mechanism of incisional hernia formation. Surgical strategies

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 141should therefore be developed to minimize early mechanical wound containing cells. Control cells were significantly better at cell migration

in comparison to over-expressing av integrin cells (p < 0.0001).failure perhaps by accelerating the recovery of fascial wound strength.Transfected dermal fibroblasts over-expressing av integrin are impaired

12. at FPCL contraction and show a reduction in cell locomotion. Thesesame characteristics were noted in fibroblasts derived from the patientwith the aberrant scar. It is proposed that the over-expression of �vintegrin alters scar maturation and collagen organization.

14.

GENE ARRAY ANALYSIS OF THE IMPACT OF AGING AND ISCHEMIAON WOUND HEALING IN RATS.

Mark Sisco, Jon E. Mogford, Alan Robinson, Yanan Zhao, Thomas A.Mustoe.Wound Healing Research Laboratory, Div. of Plastic Surgery,Northwestern University Medical School, Chicago, IL.

Research has shown that persistent tissue ischemia and aging areindependent features that severely impair the healing of chronicwounds. The compounding effects of localized ischemia and advancedage on wound healing, as measured by the formation of granulationtissue and neo-epithelium, have been demonstrated by our laboratoryusing a rat ear dermal ulcer model. However, the pathophysiology ofthese processes as they relate to chronic wounds is poorly understood.We performed gene-array analysis of tissue from wounds in a effortto elucidate the effects of ischemic stress on the wound healingresponse in the aged. Eight aged rats (24 months old) and eight youngrats (3 months old) were used for this study. The left ear of each animalwas rendered ischemic by surgically transecting 2 of 3 arteries, afterwhich a single 6-mm full-thickness ulcer was made on the ventralaspect of each ear. The wounds were harvested at 7 days post-wounding. One half of each wound, along with 1 mm of the surroundingconcentric tissue, was used for mRNA extraction from which aradiolabelled cDNA library was made using a gene-specific primer mix.This cDNA was hybridized to a nylon membrane on which 1176 cDNAsspecific to the rat were spotted. The relative intensities of digitizedphosphorimages were analyzed on a computer using array-specificsoftware. A twofold difference was considered significant. Preliminaryglobal analysis of gene expression suggests that ischemia engendersa markedly different gene expression profile in aged animals comparedto young animals. Importantly, all possible alterations in gene13.expression are found in samples from aged ischemic wounds whenTHE OVEREXPRESSION OF �V INTEGRIN BY HUMANcompared to nonischemic samples. Specifically, expression of certainFIBROBLASTS ESTABLISHES A CELL PHYSIOLOGY THAT MIMICSgenes is up-regulated in the ischemic environment indicating that geneA FORM OF ABERRANT SCARRING.regulation is not simply depressed by advanced age.

*Howard Levinson, ‡James E. Hopper, *Gregory C. Saggers, *DonaldR. Mackay, *H. Paul Ehrlich. *Section of Plastic and Reconstructive

15.Surgery and ‡ Department of Biochemistry and Molecular Biology, A BIODEGRADABLE WOUND DRESSING FOR LARGE SURFACEPenn State University, M.S. Hershey Medical Center, Hershey, PA. WOUNDS.

The increased expression of av integrin in fibroblasts derived from a CAW Juergens1, I Burkard2, H Hierlemann3, T Krause1.Trauma and Reconstructive Surgery1, BG-Trauma Center, Hamburg;patient with abnormal scarring has been reported. The patient

developed a weak, stretched scar and fibroblasts grown out from that Merck Biomaterial GmbH2, Biomet Merck; ITV Denkendorf3.scar were found to over-express av integrin. Compared to his normaldermal fibroblasts, his scar fibroblasts were poor at fibroblast a : Removal of temporary dressings used for treatment of large surface

wounds is often painful, traumatic or increases the risk of infection.populated collagen lattice (FPCL) contraction. Our hypothesis is over-expression of �v integrin in dermal fibroblasts retards free-floating For this reason we developed a biodegradable dressing that does not

need to be changed until definite wound closure.FPCL contraction and inhibits cell migration. A cell line of normalhuman dermal fibroblasts (HF) was derived from a discarded foreskin.

b : Copolymers of lactic and caproic acid were moulded to a transparentHF were transfected with an av integrin expression vector (�v-3.1 �pcDNA) leading to over-expression of av integrin. Control HF were film and physical properties were adapted to the needs of a dressing

in terms of tensile strength, flexibility and permeability. Changes oftransfected with the same expression vector (3.1 � pcDNA) lackingthe �v cDNA. Both cell lines were incorporated into FPCLs with an the material by degradation and sterilization procedures were

examined. The suitability for clinical use was proved by toxicologicalinitial diameter of 16 mm. FPCLs were composed of 25,000 cells, 0.6mg of acid soluble rat tail tendon collagen, and culture medium and bacteriological tests and in animal trials on the rat in comparison

with the nondegradable Opsite� film. Clinical application on split-skinsupplemented with 10% fetal bovine serum contained in a total volumeof 0.5 ml. FPCL contraction was measured daily for 3 days. Cell donor sites and noninfected wounds was evaluated by permission of

the Ethics Commission.morphology in FPCL used phase contrast microscopy, rhodamine-phalloidin staining of microfilaments and immuno-staining for av

c : The pH measurements on the film surface during hydrolysis in vitrointegrin. A cell migration assay compared cell locomotion betweencontrol and the over-expressing cells. Control FPCLs contracted faster show a decrease from 5.4 to 3.6. In the animal trials there was also a

clear shift to lower pH values on the wound base as compared toand four-fold greater in area than FPCLs containing over-expressingfibroblasts (n � 16). By phase contrast microscopy, over-expressing Opsite�. Probably due to of this acidity, bacterial colonies on agar

plates grew markedly slower underneath the polymer film than thosecells in FPCLs appeared less elongated than controls. The organizationof cytoplasmic microfilaments was similar in both cell lines. There was beside the film sheet or under Opsite�. Toxicological examinations

with the MTT test and keratinocyte cultivation on polymer sheetsa greater density of av integrin staining in over-expressing av integrin

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001142 Meeting Schedule and Abstracts

indicated no cytotoxic effects. In the animal trials the rats showed no sharp excisional debridement, HSE was placed on to the woundutilizing interrupted 4-0 or 5-0 absorbable sutures with a one-millimeterrestriction of mobility or pain reactions even if the sheet was touched

or removed. Granulation tissue was less cell-rich, capillarization started distance between the newly debrided skin edge and the cut HSE (figureB). Healing was defined as 100% epithelialization and the absence ofearlier and was more intense than under Opsite�. Clinical application

was performed on 80 split-skin donor sites and on 20 acute wounds. drainage. Thirty of the 47 (74%) patients with chronic wounds wenton to 100% healing. Seventy-three out of 74 wounds that healed requiredThe average wound area was 95 cm’’ (20 - 608 cm’’). Complications

were leakage in 4 cases and 1 local infection. With the use of a polymer only one or two applications of HSE to heal. The application of HSEwith the surgical principles of a traditional skin graft results in theglue there was seen moderate exudate retention but no local irritation.

Dressing changes were not neccessary, there was no molestation by accelerated healing of chronic wounds. Debridement of all scar andinfection may decrease the applications necessary of HSE that ispain (algometry 1-6 � 1,52) and nursing intensity was very low.required for successful healing and accelerated healing of chronicwounds.d : The advantages of this degradable wound dressing are first, the

fact that no removal is necessary, with a decrease of secondarycontamination and nursing intensity and an increase of patient comfort. 18.

Second, the influence on wound healing by a moist environment, USE OF ACELLULAR HUMAN DERMIS AS A CARRIER MATRIX FORimpermeability for bacteria and an acid environment. Moreover the ADENOVIRAL-MEDIATED GENE TRANSFER.dressing could be used as a drug delivery system by integration of

A. Gordon, J. Karmacharya, G. Ong, O. Hunenko, M. Esteves, B. Martin,pharmacologically active substances.T.M. Crombleholme, and R.E. Kirschner. The Children’s Institute forSurgical Science, The Children’s Hospital of Philadelphia, Philadelphia,

16.PA.FIBRIN SEALANT TISSEEL� IS A BIOCOMBATIBLE POLYMER FOR

HUMAN-DERIVED MONOCYTES, FIBROBLASTS ANDThe extracellular matrix has been shown to play a critical role in theKERATINOCYTES.cellular events necessary for tissue repair. We hypothesize that deliveryof adenoviral constructs designed to promote growth factor synthesisMarietta Cole, Steve Cox, Jon Mogford*, Miyeko Mana, Wendy Ho,

Ammie Eggleston, Thomas Mustoe* and Nabil Tawil. from an intact extracellular matrix will result in improved woundhealing. The specific aims of these experiments were designed to* Wound Healing Research Laboratory, Northwestern University,

Chicago, IL. Wound Mgt. Group, Baxter Healthcare Corp, Duarte, CA. examine the ability of acellular human dermis to serve as an effectivecarrier and to provide sustained delivery of adenoviral vectors both

Fibrin sealant products such as Tisseel� are used mainly for in-vitro and in-vivo settings. In vitro experiments: Acellular dermalgrafts (LifeCell Corp., Woodlands, TX) measuring 8 mm in diameterhemostasis. There is an interest, however, to use Tisseel to treat acute

and chronic wounds. Since the clots formed by Tisseel� contain a were incubated for one hour in a solution containing an E1-deletedCMV-driven adenovirus carrying E.Coli-derived beta galactosidase genehigher concentration of fibrinogen (50 mg/ml) and thrombin (250 U/

ml) than that seen in naturally formed fibrin clots, we are interested LacZ (AdLacZ; 5x108 PFU’s/ml). To assess efficiency of vector delivery,the dermal grafts were then co-cultured for 24 hours with a series ofin examining the behavior of cells known to be present during the

wound healing process, such as monocytes, fibroblasts and fresh human fibroblast cultures every 24 hours for one week. Thefibroblasts were stained using X-gal 72 hours after removal of thekeratinocytes, with the clots formed by Tisseel�. We found that human-

derived monocytes, fibroblasts and keratinocytes adhered and dermal carriers. In vivo experiments: Two 8 mm diameter wounds weremade on the dorsal surface of six diabetic mice, C57Bl/KsJ-db/db,proliferated well on clots formed by Tisseel�. The data also indicate

that up to 53 different genes in monocytes and about 200 genes in preserving the panniculus carnosus. In 3 db/db mice (Group I), onewound was untreated, while the other was treated with an 8mmfibroblasts were regulated in cells seeded on the clots for one day

when compared to cells seeded on a culture dish. Moreover, a different diameter, 0.001 inch thick acellular human dermal graft. In theremaining 3 animals (Groups II), the wound was treated with the dermalset of genes was regulated depending on the cell type and on the length

of time cells were seeded on clots. In addition, the cell surface graft pretreated by one-hour incubation in a solution with E1-deletedCMV-driven adenovirus carrying E.Coli-derived beta-galactosidaseexpression of integrins and growth factor receptors is altered in cells

growing on Tisseel� clots when compared to those growing on a reporter gene LacZ (AdLacZ; 5x108 PFU’s/ml). The wounds wereharvested in seven days and stained using X-gal. In vitro dermal graftsculture dish. Furthermore, we found that cells from the periphery of

wounds invaded the clots added to linear incisions created on the back effectively continued to transfect the fibroblasts for 7 days. In theanimal model, the acellular dermal matrix demonstrated enhancedof rats. The above data suggest that fibrin sealant Tisseel� presents

a biocombatible surface for cells to grow on. The data also support epithelialization and cellular infiltration with no evidence of graftrejection. The connective tissue cells surrounding the acellular dermalthe use of Tisseel to temporarily fill the acute and chronic wounds,

which are eventually, are invaded by endogenous cells replacing Tisseel graft were transfected up to 7 days. Acellular human dermis caneffectively serve as a scaffolding for sustained adenoviral vectorclots and forming new tissues.delivery for gene therapy in wound healing.

17.

SURGICAL TECHNIQUES AND HEALING IN THE UTILIZATION OF 19.

HISTOLOGIC FINDINGS IN CHRONIC WOUNDS GRAFTED WITH148 CONSECUTIVE SKIN GRAFTS WITH BIOENGINEERED HUMANSKIN EQUIVALENT IN PATIENTS WITH CHRONIC WOUNDS. BIOENGINEERED SKIN.

Brem H, Balledux J, Kaplan D, Hollier L. Mount Sinai Medical Center, E. V. Badiavas, Janet Butmarc, and Vincent Falanga. Department ofDermatology and Skin Surgery, Roger Williams Medical center, BostonNew York, NY.University School of Medicine.

Deep debridement and the creation of new wound edges will converta chronic wound into an acute wound resulting in an optimized wound Graftskin, an organotypic culture of skin derived from human neonatal

keratinocytes layered over human neonatal fibroblasts embedded in abed preparation. Human Skin Equivalent (HSE) is a bilayered, livingskin construct consisting of fibroblasts and keratinocytes with the collagen matrix, has recently been approved for the treatment of

chronic venous and diabetic wounds. In a randomized controlledsame surgical principles as a traditional skin graft. Over a 14-monthperiod, we prospectively treated 47 patients with 1) venous stasis ulcers clinical trial of 293 patients (Falanga et al, 1998) no significant adverse

effects and no obvious signs of clinical rejection were reported inof greater than one year in duration, 2) surface diabetic foot ulcers,and 3) pressure ulcers (sacral, trochanteric and ischial), with patients receiving graftskin. In vitro testing of patients treated with

graftskin revealed no evidence of specific cellular or humoral immunitydebridement and HSE. Ninety-one chronic wounds received 148 grafts.Patients with known osteomyelitis bone exposed under the wound or to graftskin. Histologic analysis of human wounds treated with graftskin

has however been quite limited. Here we report our preliminarywounds of other etiologies were not included. The diameters of thewounds ranged from 0.7 cm to 32 cm. The wounds were debrided; all histologic observations in a cohort of 14 patients with 17 wounds

treated with graftskin in whom biopsies of the grafted site were takenscar and grossly infected tissue were removed. Symmetrical slits weremade in the new skin graft, HSE, (figure A) with a #10 blade. After at least 2 weeks after application of graftskin. The etiology of these

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 143

Based on previous work, FGF-1 was selected as the angiogenic agentulcers included wounds due to arterial and/or venous disease, anextensive poorly healing burn wound and a poorly healing surgical for skin applications. For the matrix, both fibrin and albumin have

been selected for their adhesive properties as well as ability to servewound. Thickening of the grafted bioengineered skin was seen in allsamples where residual graftskin could be identified. Mucin deposition as a degradable drug delivery system. The clinical efficacy of the fibrin/

FGF-1 system has been evaluated in a burn study. The fibrin was usedwas noted in the dermal layer of both the wounds and graftskin inmost of the specimens examined. Graft take tended to occur in wounds for skin graft attachment and the wounds on the patients were

randomized (double blinded) into two groups: (1) fibrin with FGF-1that were extensively surgically debrided prior to graftskin application.Myoblast proliferation was also noted in these extensively debrided and (2) fibrin without FGF-1. Each patient also has a corresponding

control (stapled or sutured graft) for comparison.wounds. Unexpectedly, and in spite of good clinical outcome, 4 of the15 specimens exhibited a foreign body like granulomatous response.

Preliminary results have indicated that the fibrin systems keep theThese early histological observations suggest that stimulatoryinteractions develop between graftskin and the wound. blood perfusion level at a higher level longer than the controls. This

corresponds to earlier epithelialization and less graft stiffness. Formeshed skin grafts, the vascularity is kept higher, than controls, until20.the mesh fills in. This is believed to be associated with increased newtissue formation, quicker epithelialization, and less scarring. Thealbumin system is currently being optimized for adhesive strength,porosity, and degradation rate; prior to addition of an angiogeic factor.

This work was supported by CDC-NCIPC and the UAB ICRC.

22.

HUMAN KERATINOCYTES TRANSDUCED WITH THE �3 INTEGRINSUBUNIT cDNA CAN ADHERE TO GELATIN.

Miyoko Kubo, Richard A.F. Clark. Lorne Taichman, and TakahikoMoriguchi. Department of Plastic and Reconstructive Surgery,Kawasaki Medical School, Kurashiki, Japan, and Department ofDermatology, School of Medicine, and Oral Biology and Pathology,School of Dental Medicine, SUNY at Stony Brook, Stony Brook, NY.

During the re-epithelialization of cutaneous wounds, migratingepidermal cells up-regulate integrins a5�1, av�5, av�6 but no av�3.av�3 is a multi-ligand integrin receptor which interact with fibrin(ogen),fibronectin, vitronectin, and gelatin. We previously reported thatcultured normal human keratinocytes do not express av�3 on the cellsurface as in vivo keratinocytes, and do not adhere to and migrate onfibrin(ogen). Furthermore, we reported that human keratinocytestransduced with the �3 integrin subunit cDNA by a retrovirus-mediatedtransduction method express av�3 on the cell surface and adhere tofibrin(ogen), fibronectin and vitronectin significantly compared withb-Gal cDNA-transduced keratinocytes (control). In this study weexamined whether these �3 cDNA-transduced keratinocytes adhere togelatin using 1 hour cell adhesion assay. �3 cDNA- transducedkeratinocytes adhered to gelatin (around 30% total), whereas nosignificant adhesion was observed with control cells (�-Gal cDNA-transduced keratinocytes and normal human keratinocytes). Theadhesion to gelatin was inhibited by LM609 (monoclonal antibody toavb3) and RGD peptides in a dose dependent fashion but not by normalmouse IgG nor RGE peptides. These data indicate that humankeratinocytes transduced with the b3 cDNA can adhere to denaturedcollagen as well as fibrin(ogen). And these dada support an idea thatthese recombinant keratinocytes provide a method of enhancing woundhealing in a graft procedure when they are applied to fibrin(ogen) and

21. denatured collagen.OPTIMIZING TISSUE ADHESIVE SCAFFOLDS FOR SKIN WOUNDS.

23.S. Range, T. Barker, B. Blum, D. Kilpadi, R. Overby, and D. Feldman. MORPHOLOGICAL EVIDENCE IN SUPPORT FOR THE ROLLINGDepartment of Biomedical Engineering, University of Alabama at MODEL OF HUMAN EPIDERMAL REPAIR.Birmingham, Birmingham, AL.

Marcia Usui, Jonathan Mansbridge, Lara Muffley, Jerrie Larsen andJohn Olerud.To determine the optimum system for each clinical application the

migration rates of key cells, are important. For example, fibroblasts Department of Medicine (Dermatology), University of Washington,Seattle, WA., Advanced Tissue Sciences Inc., La Jolla, CA., Veteransneed to be within 0.1 mm of the blood supply to have sufficient oxygen

to produce collagen as well as migrate up to 0.2 mm/day. Therefore Affairs Puget Sound Health Care System, Seattle, WA.angiogenesis becomes the rate-limiting step. The speed of angiogenesisalso determines the earliest that the matrix can support epidermal Our studies have focused on the role of keratinocyte migration in re-

epithelialization of human cutaneous wounds. The most well-cells. Therefore, strategies to speed up angiogenesis are needed. Thiswould include use of angiogenic growth factors or modification in the documented model (the ‘‘sliding model’’) focuses on the role of basal

keratinocytes as they undergo cell shape change, release theirmatrix configuration (e.g. porosity or thickness) to assure thatangiogenesis was not the rate-limiting step. With natural biomaterials, hemidesmosomal anchorage to the basement membrane, enter a

migratory mode and laterally migrate across the provisional woundsuch as albumin or fibrin, delivery of an angiogenic factor can beaccomplished in a number of different ways. If the factor is bound to matrix. Extensive in vitro studies and studies of simple epithelia

support this ‘‘sliding model’’. A less well-characterized model (thethe matrix, it is released only when the natural material degrades.Therefore, the release is controlled by the rate of phagocytic cellular ‘‘rolling model’’) describes re-epithelialization of complex stratified

epithelia. In the ‘‘rolling model’’, basal keratinocytes remain firmlyinfiltration, and thus under biofeedback control.

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001144 Meeting Schedule and Abstracts

attached to the basement membrane while the suprabasal keratinocytes 26.

EPIDERMAL GROWTH FACTOR RECEPTOR OVEREXPRESSIONroll or leap frog over the basal keratinocytes. Normal incisional woundswere created on volunteers using Simplate-II bleeding time devices at INDUCES DISSOLUTION OF CELL CONTACTS AND DOWN-

REGULATION OF JUNCTIONAL PROTEINS.1, 6, 24, and 72 hrs prior to biopsy. Wound epithelia were evaluatedfor K10 and K2e expression using standard immunohistochemistry.

Jennifer Halbleib, Dominic Ortega and Laurie Hudson. University ofResults from these morphological studies show positive K10 and K2eNew Mexico Health Sciences Center, Albuquerque, NM.staining keratinocytes in the migrating epithelium including cells in

direct contact with the provisional matrix. The two keratins, knownReepithelialization is required for wound repair and restoration ofto be markers of epidermal differentiation, are normally expressed byepidermal barrier function. Desmosomes disappear during thesuprabasal keratinocytes. Their presence on the migrating epithelialmigratory phase of reepithelialization and desmosome reassemblytongue appears to support of the ‘‘rolling model’’ of epidermal repair.marks the end of keratinocyte migration; however, the mechanismsunderlying desmosome assembly and disassembly are unknown. A role24.for the epidermal growth factor receptor (EGFR) in wound healingIMMUNOLOCALIZATION OF TENASCIN-C, ALPHA9 INTEGRIN ANDhas been proposed and there is evidence that EGFR and its ligandsALPHAVBETA6 INTEGRIN DURING WOUND HEALING IN HUMANare dynamically and transiently upregulated during reepithelialization.ORAL MUCOSA.In this study, we examined the ligand-dependent effects of EGFR

Zhenming Fan; Wen-Bin Ho*; Xinfan Huang**. overexpression on junctional stability in a keratinocyte cell line. SCCFaculty of Dentistry, Health School of Yiyang, Hunan, PRC. *Vivotech, 13 cells were transfected with either vector alone (SCC 13 vector) orInc. Mountain View, CA. ** Connetics Corp. Palo Alto, CA. the EGFR gene (SCC 13 EGFR). The SCC 13 vector line expressed

EGFR levels equivalent to normal keratinocytes, while there was anTenascin-C (TN-C) and its isoforms are multidomain extracellular approximate five-fold increase in EGFR expression in the SCC 13 EGFRmatrix proteins that are believed to be involved in the regulation of cells. EGF-dependent colony dispersion was observed in the SCC 13stromal-epithelial interaction. Some of the interactions between TN-C EGFR cells. Neither increased EGF concentration nor extended EGFand cells are mediated by integrins. In this study we analyzed the treatment promoted this response in the SCC 13 vector cells.expression of TN-C and the putative TN-C binding alpha9 and Furthermore, an EGF-dependent disruption of desmosomal junctionsalphavbeta6 integrins during human wound healing using was only detected in the SCC 13 EGFR cell line. Upon treatment withimmunohistochemical staining. In 3-day-old oral mucosal wounds, EGF, a subset of junctional proteins were down regulated includingimmunoreactivity for alpha9 integrin localized abundantly at the plakoglobin (gamma-catenin), beta-catenin, and, to a lesser extent, E-migrating basal wound epithelial cells. TN-C was localized in the matrix cadherin. However, the levels of desmosomal components desmoplakinbetween and underneath alpha9-expressing epithelial cells. In parallel and desmoglein were unchanged. Since it is known that growth factorwith gradual downregulation of alpha9 integrin immunoreactivity in 7- stimulation results in plakoglobin relocalization from cell junctions today and older wounds, the expression of alphavbeta6 integrin was the cytoplasmic compartment, the levels of plakoglobin in celltemporarily induced. Integrin alphavbeta6 co-localized in the same area compartments were studied using subcellular fractionation. The mostas TN-C at the cell-cell contacts of the basal and suprabasal cell layers dramatic difference in plakoglobin protein between the two cell linesof the wound epithelium. During granulation tissue formation and was found in the cytoskeleton-associated pool. Junction-associatedreorganization from 7 to 28 days after wounding, TN-C is expressed plakoglobin was nearly eliminated in the SCC 13 EGFR cells. Theseunder the migrating epithelial front and in the granulation tissue during findings indicate that EGFR expression levels play an important rolematrix deposition in wound repair. Preferential localization of alpha9 in junctional protein localization and expression and suggest that EGF-integrin in migrating epithelial cells and of alphavbeta6 integrin in dependent responses are modulated not only by the concentration andepithelium after wound closure suggests different functions for these availability of ligand, but additionally at the level of receptor abundance.integrins in wound repair.

27.25.IP-9 AS A MEDIATOR OF DERMAL AND EPIDERMALLIFESPAN EXTENSION AND RESTORATION OF WOUND HEALINGCOMMUNICATION DURING WOUND REPAIR.BY TELOMERASE IN AGING KERATINOCYTES.

Latha Satish, Angela Glading, Dorne Yager*, Alan Wells. DepartmentXu-Rong Jiang, Adrienne Bronstein, Donghee Choi, Alivelu Irrinki,Choy-Pik Chiu, Allison C. Chin and Calvin B. Harley. Geron Corporation, of Pathology, University of Pittsburgh, Pittsburgh, PA 15261 and

*Medical College of Virginia, Richmond, VA 23298.Menlo Park, CA.

Normal wound healing is a complex, highly regulated dynamic process,Most normal human somatic cells possess low or undetectable levelsof telomerase and their telomeres shorten with each cell division, which requires co-ordinate response of both epidermal and dermal

compartments. To accomplish the healing process several growthultimately leading to replicative senescence. Human skin provides avaluable model to investigate the potential role of telomerase in chronic factors, chemokines and matrix elements signal during the

inflammatory, reparative and resolving phases. We have found that thedisease. We have previously demonstrated that ectopic expression oftelomerase immortalizes dermal skin fibroblasts and microvascular ELR- CXC chemokines IP-10, Mig and PF4 limit fibroblast

responsiveness to growth factors. The purpose of the study is toendothelial cells while maintaining growth control and differentiatedfunction. However, the third major skin cell, the keratinocyte, is determine (a) whether the keratinocyte-derived member of this ELR-

CXC family, IP-9 (also known as I-TAC, beta-R1, H-174) similarly inhibitsreportedly refractory to telomerase immortalization unless the pRb/p16INK4a pathway is inactivated. Since keratinocyte proliferation is fibroblast responsiveness and (b) if IP-9 affects keratinocyte

functioning during wound repair. We hypothesize that IP-9 signal toan important component of normal skin turnover and wound healing,and loss of telomeres, proliferative capacity, and function are the dermal compartment to synchronize the re-epithelialisation

process. We found IP-9 to be expressed by keratinocytes at the marginsassociated with skin aging and chronic wounds, we sought toreinvestigate the ability of telomerase to extend the lifespan and of human wounds and to be induced by mechanical wounding of

keratinocyte monolayers. Our studies show that the IP-9 limits theyouthful function of human keratinocytes. Here we report that underappropriate culture conditions, ectopic hTERT expression alone can growth factor induced fibroblast motility to 43%, similar to other ELR-

negative CXC chemokines IP-10 (55%), PF-4 (45%) and MIG (49%) asimmortalize human keratinocytes. The lifespan extended and hTERT-expressing keratinocytes retain p16INK4a, pRb and p53 protein determined in a two dimensional in-vitro wound healing assay. In

contrast, IP-9 stimulated the motility of undifferentiated keratinocytesexpression, respond to normal growth control, differentiate in responseto high cell density, growth factor withdrawal and phorbol ester, and by 29% and had no effect on EGF- induced motility. IP-9 also increased

calpain activity. This increase in basal motility and calpain activitydo not spontaneously activate c-myc. Further, both stable and short-term telomerase expression in keratinocytes can accelerate healing in could be blocked by inhibitors of PLC and Mu-calpain, suggesting that

this requires a PLCbeta - calcium flux - Mu-calpain signaling pathway.a tissue culture wound model. Thus, telomerase is sufficient for lifespanextension in human keratinocytes and may prove useful in the Interestingly, IP-9 increased EGF-induced proliferation of

undifferentiated keratinocytes (143% compared to 100% for EGF alone)treatment of chronic wounds where keratinocyte function iscompromised. but had no effect on the basal or EGF-induced proliferation of

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 145differentiated keratinocytes. The above data suggest a model that IP- 30.

THE KINETIC CONTRIBUTION OF BONE MARROW-DERIVED CELLS9 alters the functions and behavior of fibroblasts and keratinocytesduring wound healing. TO HEALING CUTANEOUS WOUNDS.

Susan R. Opalenik and Jeffrey M. Davidson. Department of Pathology,28. Vanderbilt University, Nashville, TN.SPARSELY PLATED KERATINOCYTES SPONTANEOUSLY

APOPTOSE IN A NITRIC OXIDE-LIMITED MANNER. The origin of the wound fibroblast has been historically attributed tothe migration of cells from the local tissue environment to the woundR Weller, T Billiar, Y Vodovotz. Department of Surgery, University of

Pittsburgh Medical Center, PA. site. Exciting new evidence suggests that the bone marrow (BM)compartment houses stem cells with self-renewal and differentiation

Sparsely plated keratinocytes, not exposed to contact inhibition, divide capacities for not only cells of the hematopoietic system, but also cellswith mesenchymal phenotype. The aim of this study was to determinerapidly, in common with basal keratinocytes. However, sparsely

populated cells are also known to undergo apoptosis in the absence the kinetic and quantitative contribution of BM-derived cells to healing,cutaneous wounds. Sex-mismatched, bone marrow transplantation wasof trophic signals from adjacent cells. Nitric oxide is anti apoptotic,

and present in human skin both from surface reduction of sweat nitrate, performed utilizing a novel transgenic mouse line which houses theluciferase and beta-galactosidase marker genes under theas well as due to the activity of constitutive NO synthase in

keratinocytes. We studied the effects of plating density and nitric oxide transcriptional control of the enhancer/promoter of the mouse type-Icollagen alpha-2 chain. Excisional, cutaneous wounds were generated(NO) on spontaneous apoptosis.on stable chimeras and control mice by punch biopsy. At defined time-points, wounds were re-excised and tissue processed for DNA/RNAHuman keratinocytes (CCD1106) were plated on 6 well dishes at a

density of 1.7, 3.4 and 6.8 � 104 cells per cm2 . 48 hours after plating, isolation. Real-time PCR was performed with male-specific (SRY)primers to determine the kinetic and quantitative contribution of BM-apoptosis was measured by flow cytometry assessing binding to

annexin V. Spontaneous apoptosis was 10.2, 9.6 and 4.4% respectively. derived cells to the wound environment. Moreover, luciferase-specificprimers were utilized in quantitative RT-PCR to examine the kineticIn a separate experiment, addition of the NOS antagonist, L-NAME, to

cells plated at the lowest density increased spontaneous apoptosis activation of the type-I collagen alpha-2 chain enhancer/promoter. Ourobservations demonstrate the migration of transplantable, BM-derivedafter 24 hours from 2% to 19%, and this was reduced to 1.5% by the

addition of 10uM of the NO donor SNAP. Culturing keratinocytes in cells to cutaneous wounds where they remain part of the remodeledtissue at least 18days post-injury. Moreover, these cells activate thethe presence of 200uM of the Caspase 3 inhibitor Ac-DEVD-cho also

abrogated spontaneous apoptosis. collagen type-I promoter, suggesting a mesenchymal differentiationprogram. Future studies will focus on elucidating the specific cellular

Proliferating basal keratinocytes are exposed to pro and anti-apoptotic phenotype of BM-derived cells within the wound environment and theirultimate contribution to the resto.signals, which they must integrate to determine their fate: proliferation,

terminal differentiation, or apoptosis. We have demonstrated theextreme sensitivity of dividing keratinocytes to the anti-apoptotic 31.

DELAYED WOUND HEALING IN MAC-1 (CD11b/CD18) INTEGRINeffects of spontaneously produced NO, and believe that this opposesan innate tendency to enter the apoptotic pathway. These findings may KNOCKOUT MICE.indicate a novel role for endogenous NO production in wound healing. Mark Sisco, Jerome Chao, Injoong Kim, Tanya Mayadas-Norton, Jon

E. Mogford, Thomas A. Mustoe. Wound Healing Research Laboratory,29. Div. of Plastic Surgery, Northwestern University Medical School,THE CALCIUM-BINDING PROTEINS S100A8 AND S100A9 ARE Chicago, IL.ENCODED BY NOVEL INJURY-REGULATED GENES.

The Mac-1 integrin is an important mediator of migration andIrmgard S. Thorey (1), Johannes Roth (2), Johannes Regenbogen (3),Andreas Goppelt (3) and Sabine Werner (1). inflammatory activation of neutrophils and monocytes. However, the

role of Mac-1 in modulating macrophage emigration and activation and(1) Institute of Cell Biology, ETH Zurich, Switzerland. (2) Institute ofExperimental Dermatology, University of Muenster, Germany. (3) its subsequent impact on cutaneous wound healing have not been well

studied. To examine the significance of Mac-1 to murine wound healing,SWITCH Biotech AG, Martinsried, Germany.we measured epithelialization and granulation tissue formation inpartial-thickness ear wounds and full-thickness head wounds,To gain insight into the molecular mechanisms underlying cutaneous

wound repair, we performed a large scale screen to identify novel respectively. Superficial 3-mm wounds were made on the ears of Mac-1 knockout (n � 5) and control (n � 7) mice. Full-thickness 5-mmgenes involved in this process. Here we show that, following cutaneous

injury in mice, RNA and protein levels of the two Ca2�-binding proteins wounds were made on the skulls of Mac-1 knockout (n � 6) andcontrol (n � 7) mice. Wounds were histologically analyzed at post-S100A8 and S100A9 are strongly upregulated in the hyperproliferative

epithelium at the wound edge. Furthermore, expression of both wounding days (POD) 3, 5, and 7. The gap measured between theleading edges of inward migrating granulation tissue was significantlyproteins is induced in acute and chronic human wounds. It has been

previously known that S100A8 and S100A9, while being absent from increased in knockout mice compared to control animals at day 5 (3.8� 0.3 mm vs. 2.6 � 0.5 mm; p < 0.001) and at POD 7 (2.2 � 0.4 vs.normal skin, are abundant in the lesional keratinocytes of patients

suffering from inflammatory and hyperproliferative skin diseases, but 0.96 � 0.73 mm; p � 0.005). Epithelial gap measurements were alsoincreased in knockout mice versus wild-type controls at days 3 and 5it is not clear whether this induction is related to the inflammation or

the hyperproliferative state of the epidermis in these conditions. (0.62 � 0.02 vs. 0.54 � 0.07 mm, p < 0.02 and 0.58 � 0.06 vs. 0.39 �0.08 mm, p < 0.001, respectively). These findings suggest that functionalTherefore, we analyzed S100A8 and S100A9 expression in the

hyperthickened but not inflamed epidermis of transgenic mice Mac-1 plays an important role in cutaneous wound healing, and thatits absence retards both the deposition of granulation tissue and woundoverexpressing activin from a basal keratinocyte-specific promoter and

found both RNA and protein expressed at elevated levels. From this epithelialization.we conclude (1) that S100A8 and S100A9 are novel players in woundrepair where they might be involved in the regulation of inflammationand/or keratinocyte differentiation; and (2) that upregulation of S100A8and S100A9 can occur in the absence of inflammation.

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001146 Meeting Schedule and Abstracts

32. of the MRP8 subunit results in a marked change in collagen synthesisin the rabbit ear model. Experiments with the heterodimeric complexMODULATION OF MATRIX METALLOPROTEINASE 2

BIOAVAILABILITY DURING WOUND HEALING BY THE are underway.ANGIOGENESIS INHIBITOR, THROMBOSPONDIN 2.

Themis R. Kyriakides, Azin Agah, Grace Huynh, and Paul Bornstein. 34.University of Washington, Seattle, WA. LONG-TERM HEALING OF VENOUS STASIS ULCERS TREATED

WITH SERUM-FREE CULTURED EPIDERMAL AUTOGRAFTS.Excisional wounds in mice that lack the potent angiogenesis inhibitor,

J. Burdge (1), F. Cope (2), B. Abbruzzese (2) , and J. Wille (2).thrombospondin (TSP) 2, heal at an accelerated rate and resolve with(1) Wound Care Center, Riverside Hospital, Columbus, OH andminimal scarring. Previously, we observed that such wounds displayed(2) Hy-Gene Biomedical Corporation, Columbus, OH and Trenton, NJ.prolonged neovascularization and abnormal extracellular matrix

deposition. In this study we investigated whether the reported abilityPreviously, we reported that cultured epidermal autograft (CEA) sheets,of TSP2 to modulate activation of TGF-beta1 and the bioavailabilty ofproduced by a novel serum-free culture and tissue engineeringmatrix metalloproteinase (MMP)-2 played a role in healing. In addition,technology (Autoderm&#8482;; Hy-Gene Biomedical Corporation),we analyzed the spatiotemporal deposition of TSP1 and TSP2 in thewere applied to recalcitrant venous stasis ulcers to determine if thiswound bed. Full-thickness excisional wounds were made with the aidclinical approach would be efficacious and cost-effective. A total ofof a 6-mm biopsy punch and harvested on days 3, 7, 10, and 14 post15 patients with full-thickness venous stasis ulceration withoutwounding. Immunohistochemical analysis revealed that TSP1 and TSP2infection were assigned to weekly (for 8 weeks with followup at 12have distinct non-overlapping deposition patterns, with the early (dayweeks) debridement, high compression multilayered wrapping, and the3) phase of the response dominated by TSP1 and the late (day 10) byapplication of the CEA (group � AAG; n � 10) or the control withoutTSP2. Furthermore, TSP1 deposition was not altered in TSP2-nullthe CEA (standard of care group, SCG; n � 5). The relative rate ofwounds, suggesting the absence of a compensatory mechanism.wound closure for AAG was significantly better (regression byRelative levels of active TGF-beta1 and MMP-2, reflecting the extentchronological group � p < 0.001; Mann-Whitney non-parametric � pof their distribution, were evaluated by histomorphometry following< 0.003) than the SCG. AAG also had a significantly higher healing rateimmunohistochemical analysis. Unlike the levels of TGF-beta1, which(8/10) than the SCG (1/5) (Kaplan-Meier � p < 0.022; Fischer’s orwere indistinguishable between control and TSP2-null wounds, theBarnard’s exact test � p < 0.04). Here, we report our finding on a 12levels of MMP-2 were significantly increased in day 10 TSP2-nullmonth followup of the patients in this study showing that in the AAGgranulation tissue. The increased MMP-2 in TSP2-null mice coincidedof the eight on-study wounds healed by the AAG three patients returnedwith maximal TSP2 expression and the onset of vascular regressionfor further wound treatment not associated with the AAG-treatedin control wounds. Therefore, we conclude that the lack of TSP2 leadswound area followed in the study. The five remaining patients did notto prolonged neovascularization of wounds due, in part, to elevatedreturn for further wound treatment of the healed area and the woundslevels of MMP-2. Moreover, TSP2 appears to play a dynamic role duringremained closed for at least one year. Of the two wounds in the AAGactive wound matrix remodeling by modulating MMP-2 bioavailabilitythat failed to heal on study, one healed at 30 weeks post enrollmentand promoting vascular regression.and the other remained unhealed at one year post enrollment. Of the

33. five patients in the SCG, the one wound which healed study remainedIDENTIFICATION OF TARGET GENES FOR THE DEVELOPMENT OF closed at 12 months post-enrollment, one additional wound healed atINNOVATIVE DRUGS TO HEAL CHRONIC WOUNDS. 21 weeks post-enrollment after placement of an autograft, and three

remained under treatment 12 months post- enrollment. Thus, at one-M. Bittner*, J.-P. Halle*, J. Regenbogen*, P. Hof*, A. Dopazo*, B.year, 90% (9/10) of the AAG CEA-treated wound had closed woundsReitmaier*, S. Werner#, J. Das Gupta@, J. Davidson@, A. Goppelt*.compared to 40% (2/ 5) of the SCG-treated wounds where one of the*SWITCH BIOTECH AG, Martinsried, Germany#, Institut furhealed SCG wounds was closed by autograft. These data suggest thatZellbiologie, ETH Honggerberg, Zurich, Switzerland,the use of the CEA produces significant long-term benefits in the@Department of Pathology, Vanderbilt Univ. Medical School Nashville,treatment of recalcitrant venous wounds.TN, U.S.A.

We have carried out extensive differential gene expression analyses 35.of murine and human wound tissue to identify genes that trigger the LOCAL WARMING INCREASES OXYGENATION AND DECREASEScomplex process of cutaneous wound healing. PAIN IN ISCHEMIC ULCERS.

By a combination of subtractive hybridisation and differential display N Puzziferri, JM West, TK Hunt, HW Hopf.University of California-San Francisco, CA.of cDNA from non-wounded skin and early murine wounds more than

100 genes have been identified, and their differential expression wasArterial insufficiency ulcers with a transcutaneous PO2 (PtcO2) < 20subsequently verified by Real Time RT-PCR. About 20% of the genes

were novel whereas 25% of the genes have previously been described mm Hg rarely heal without intervention to increase perfusion, andhence oxygenation. Warming is one of the few stimuli that canin wound healing. The remaining genes encode enzymes, enzyme

inhibitors, transcription factors, G-protein coupled receptors, ion overcome local vasoconstriction brought on by pain, hypovolemia,anxiety, and hypothermia. However, warming has traditionally beenchannels, nuclear receptors or cytokines whose function has been

described in different contexts. Candidate genes were selected for avoided for fear of increasing oxygen consumption, and thereby,worsening hypoxia.further analysis based on their expression pattern in different murine

models for impaired wound healing. Expression levels of theEight patients with documented arterial disease, and chronic stablecorresponding human homologues were quantitated in tissue of

normally healing wounds or in samples from human ulcers by a ischemic foot ulcers, were treated with local warming. Wound sizeaveraged 15cm2, and baseline PtcO2 averaged < 20mmHg(range 6-20).combination of Laser microdissection and Real time PCR. In situ

hybridisation and immunohistochemistry revealed the localization of Treatment and measurements were done in the supine position. Localwarming was applied with Warm-Up wound covers (Augustine Medical,individual gene products.Inc.; Eden Prairie, MN) at 38�C for one hour. Temperature and PtcO2were recorded. Two patients, thus far, have undergone local warmingOne of the candidate genes was identical to MRP8, a cytokine from

the S100 family which was found to be strongly induced in normal treatments for three hours/day � 12 weeks.human wounds. The spatial and temporal expression patterns of MRP8are identical to MRP14 which forms a heterodimer with the MRP8 Mean � SD skin-surface temperature was 32 � 4�C, and with warming,

rose to 33 � 4�C. In three patients there was no increase in PtcO2. Inprotein. The MRP8/14 complex is expressed in inflammatory cells andsuprabasal keratinocytes at the wound edge in various human ulcers. five of eight patients, mean PtcO2 increased to 32 � 4mmHg. In the

two patients who have undergone 12 weeks of therapy, average baselineA transient overexpression strategy by biolistic gene transfer was used PtcO2 increased from 17 � 3mmHg to 33 � 4mmHg and foot pain

levels decreased from 8 to 1 on visual analog scale.to validate target genes in vivo. The results suggest that overexpression

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 147Local warming, used judiciously, can increase oxygenation and demonstrate better outcomes with increased product awareness and

familiarity of users. This open-label prospective study supports thedecrease pain in ischemic ulcers.results of an earlier pivotal trial in that, Graftskin leads to rapid healingof long standing venous leg ulcers.36.

GRAFTSKIN IN THE TREATMENT OF DIABETIC FOOT ULCERSRESULTED IN SIGNIFICANTLY LOWER RATES OF AMPUTATIONS. 38.

THE OUTCOME OF DIABETIC LOWER EXTREMITY WOUNDSM. Sabolinski1, V. Falanga2, A. Veves3 and the Graftskin Diabetic Foot AFTER HYPERBARIC OXYGEN THERAPY (HBO2T): AUlcer Study Group. RETROSPECTIVE, MULTI CENTER ANALYSIS OF 1006 PATIENTS.1Organogenesis Inc., Canton, MA; 2Department of Dermatololgy andSkin Surgery, Boston University School of Medicine; Roger William’s C Fife, L. Smith, G Otto, R. Warriner, P Sheffield, J Mader, T Love, R.

Harrist.Medical Center, Providence, RI; 3Joslin Beth Israel Deaconess FootCenter, Boston, MA. UT Houston and Hermann Ctr for Wound Healing, Houston, TX; Otto

and Associates, Rock Hill, SC and the College of Business, Univ. ofA prospective, randomized, twenty-four center study of 208 patients North Carolina, Charlotte, Southeast TX Ctr for Wound Care, Conroe,

TX; The Nix Hyperbaric Facility, San Antonio, TX; The Dept. of Internalwas conducted to determine the safety and efficacy of graftskin incomparison to conservative treatment, saline moistened gauze, in the Medicine, The University of Texas Medical Branch at Galveston, TX,

Hyperbaric Med Ctr, Travis AFB, CA, The University of Texas Schooltreatment of diabetic foot ulcers. One hundred twelve patients receivedgraftskin and 96 received control. Safety data was obtained by of Public Health, Biostatistics, Houston, TX.spontaneous adverse event reporting over the 24-week duration of thestudy. Amputations on the study limb were compared between groups. The purpose of this study was to characterize the outcome of diabetic

lower extremity wounds after hyperbaric oxygen therapy, to identifyComplete wound closure was evaluated by 12 weeks. The rate of studywound related adverse reactions was comparable between the two predictors of benefit, and to determine the number of hyperbaric

treatments necessary to achieve a given benefit.groups except for the incidence of osteomyelitis (2.7% graftskin vs.10.4% control, p < .05) and amputations (6.3% graftskin vs. 15.6% control,

Charts of 1006 diabetic patients from 5 Texas multiplace hyperbaircp < .05). The overall incidence of complete wound closure was 56.3%for graftskin and 37.5% for control patients (p < .01). In a Kaplan-Meier chambers were retrospectively reviewed for outcome and classified

into categories: healed, partially healed, not changed, or amputated.life table analysis, median times of 65 and 90 days were shown for thegraftskin and control patients respectively (p < 0.01). At six months Patient demographics, history and treatment characteristics were

recorded in a database by a single physician observer. Data was92% of the graftskin treated patients remained healed compared to 83%for control. We conclude that graftskin may help in reducing the spread analyzed by simple cross tabulations of individual factors (chi-square),

and multiple regression. A model of outcome was created and validatedof infection to deep structures, such as bones and tendons by healingmore diabetic foot ulcers in a shorter period when compared to utilizing data from a sixth center.conservative treatments. By eliminating the open portal of entry oforganisms into the deep tissues, graftskin may be an important adjunct 76.4% of diabetic patients undergoing HBO2T had documented

improvement in the wound following a course of treatment. The averageto standard diabetic foot ulcer care in helping to reduce the incidenceof amputations. number of treatments in these patients was 34. In patients without

renal failure (RF), the factors most significantly related to outcomewere: Smoking (p � 0.004), interrupted HBO2T regimen (p � 0.000),37.

and the modified Wagner score of the worst wound(s) (p � 0.000). InGRAFTSKIN IN THE TREATMENT OF VENOUS LEG ULCERS : Aaddition, one variable was created which had predictive value: ‘‘Durage’’PROSPECTIVE STUDY.� chronological age � known duration of diabetes. Renal failure (RF)

V. Falanga, M.D, W. Pearce, L. Milstone, M.D, ES Atillasoy. Boston patients were much less likely to be helped by HBO2T than non RFUniversity School of Medicine, Roger Williams Medical Center, (57.8% vs 76.4%). The likelihood of benefiting decreased with increasingProvidence, RI, Northwestern University, Chicago, IL, Novartis modified Wagner score. The benefit rates for 635 standard care patientsPharmaceuticals Corporation, East Hanover, NJ. (no RF or growth factors) had the following relationships with modified

Wagner score: Wagner 2 (88.1%), 3 (78.7%), 4 (60%) and 5 (29.6%). BasedVenous leg ulcers (VLUs) affect approximately 2.5 million Americans on these data, a predictive model was created and validated utilizingcausing substantial disability. Graftskin is a living tissue-engineered bi- data from a sixth center. Although the R2 values of about 20% indicatelayered human skin substitute approved for treatment of VLUs that the model is not highly predictive of individual patients, it was possiblehave not adequately responded to conventional therapy. A prospective, to assess the risk of failure and estimate the likelihood of success asopen-label, multi-center study was conducted in order to assess the the number of HBO2T treatments increased.safety and efficacy of Graftskin and to quantify the minimum numberof applications required for the shortest time to healing. We conclude that it is possible to predict the likelihood of benefit from

HBO2T utilizing a mathematical model. The majority of diabeticPatients with VLUs resistant to healing after at least 4 weeks of previous wounds were improved after HBO2T. And while the likelihood of benefittreatment were enrolled. Patients returned weekly for dressing changes decreased with increasing wound severity, even some wounds withand wound assessments during the 18 week treatment period and were modified Wagner scores of 4 were improved after hyperbaric treatment.followed for 15 weeks thereafter. Hematologic studies were performedat baseline and end of study.

39.

ANALYSIS OF BECAPLERMIN ACTIVITY IN CHRONIC WOUND104 patients were enrolled and 78 patients were treated per protocol. FLUIDS FROM DIABETIC FOOT, PRESSURE, AND VENOSTASISThe mean duration of VLUs was 29.6 months, and 45.1 months in those ULCERS TREATED WITH REGRANEX�.that closed by 18 weeks. Over 90% of the ulcers that closed during thetreatment phase had surrounding skin with evidence of chronicity. An R. N. Pandey, S. Rana, K. Patel, D. Cooper*, & J. Smiell.

The R. W. Johnson Pharmaceutical Research Institute, & *Ortho McNeilaverage of 1.4 applications was needed in patients achieving 100%wound closure. The majority of those with 100% closure were healed Pharmaceutical, P. O. Box 300, Rt. 202 South, Raritan, NJ.by week 6. Of note, 3 centers with prior Graftskin experience had high,more rapid closures: 11 of 17 (65%) achieved full closure by week 18. It has been accepted that chronic wound fluid (CWF) has different

characteristics with regard to affecting proliferation of human dermalThere were no previously unreported, unexpected Graftskin relatedserious adverse events. There were no significant changes from fibroblasts, endothelial cells and keratinocytes than the acute wound

fluid. Earlier we had demonstrated that becaplermin (rh-PDGF-BB) orbaseline in blood work at the end of study.the REGRANEX� gel, following an in vivo application onto a chronicwound, maintains some of its biologic potential after 12 hours.Graftskin is safe, with no device related serious adverse events or

clinically significant hematologic changes. Complete wound closure However, no information is available about the biologic potential ofthe gel, once the gel is left on the wound beyond 12 hours.can be achieved with few applications (1.4). Future studies may

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001148 Meeting Schedule and Abstracts

The studies emphasize the importance of relieving edema to improveCWF is always contaminated with bacterial organisms and is likely tocontain endotoxins and other substances. This in vivo condition is the nutrition of tissue at risk of venous ulcers.likely to affect the integrity of becaplermin in REGRANEX�. From aclinical point of view it is important to know whether the gel is viable 41.during the application time. This information may help in determining A MODEL FOR INTEGRATING DIAGNOSTIC IMAGING INTO THEthe frequency of dressing change. Following the gel application, the TEAM APPROACH FOR WOUND HEALING.viability of becaplermin in the REGRANEX� gel was studied in diabeticfoot, pressure, and venous stasis ulcers at 12, 24, & 48 hours Moore S*, Brem H, Kapil-Pair N, Kaplan D, Hollier L. Mount Sinairespectively. The samples were analyzed by three different assays, Medical Center, Department of Radiology*, Department of Surgery,ELISA (enzyme linked immunosorbent assay), mitogenic assay and New York, NY.Western Blot.

Improved communication between radiologist and clinician, as wellBased on the wound fluid studies it is concluded that even after 24 as optimized imaging of wound complications can lead to more specificand 72 hours following the REGRANEX� gel application onto diabetic and timely treatment, accelerated healing and decreased length ofand venostasis wounds respectively, some active becaplermin was hospital stay. Magnetic resonance imaging (MRI) evaluation of woundspresent on the wound. However, in the case of pressure ulcers, not a including soft tissue infection, bone involvement and deepsingle patient showed the presence of active becaplermin in the post compartment collections or tracts was performed on patients withtreatment wound fluid. The data suggests that when using a topical several types of chronic wounds, including diabetes, sickle cell,growth factor on pressure ulcers the dressing may need to be changed pressure, and venous stasis. We have developed a wound care teammore frequently than every 48 hours for sustained becaplermin levels. model that integrates a musculoskeletal radiologist into the clinical

team to improve communication before and after imaging. The40. radiologist makes weekly bedside clinical rounds with the wound

UNRELIEVED EDEMA IS AN IMPEDIMENT TO CHRONIC WOUND healing team and the planning of the MRI scan happens in closeHEALING. communication after the radiologist has examined in the patient. The

radiologist can assist in correlating plain films with MRI findings, asRaj Mani, Lina Hammad, Geoff Roberts and Carol Collins.well as providing a better understanding of anatomic aspects of theSouthampton University Hospitals Trust, Tremona Road, Southampton,wound when the clinicians round in the radiology suite. We employEngland.electronic communications between the radiologist and the clinicalteam, drawing upon a computerized picture archiving system. TheEdema has been associated with poor healing of venous leg ulcersclinicians also provide the radiologist with the relevant history,(VLU). In studies from different centers, relieving edema was associatedinformation about the detailed area, as well as the clinical question towith improved tissue oxygen and blood flow. We report the effects ofbe answered by the specific MRI scan, on a case to case basis. In overunrelieved edema on both mechanical and microvascular properties150 wound patients, MRI has provided specific and accurate data asin tissues at risk of VLU.to the presence of soft tissue infection and presence or absence ofosteomyelitis. Close and timely communication with the radiologistDurometry was used to measure tissue stiffness. A laser Dopplerresulted in diagnoses that altered and guided treatment and outcome,flowmeter was mounted within the tabs of an extensiometer to studyincluding soft tissue infection, bone ischemia explaining nonhealingdermal blood flow changes during controlled uniaxial strain andwounds and severe pain, and anatomic details for surgical planning.relaxation. The measurements were made on patients with chronicFacilitating discussion of the MRI between radiologist and clinician,venous insufficiency (CVI) including those with frank ulcers at theas well as examination of the wound by the radiologist has increasedtime of testing, and matched elderly controls.the value of the scan in guiding treatment, utilization in evaluation forneed of antibiotics, and decreases length of hospital stay.Durometry was done on legs and forearms of subjects resting supine

in well controlled conditions (CVI group N � 30, 6M, 24F age range35-86 years mean 63.5 years, VLU group N � 14, 5M, 9F, age range 46- 42.76 years, mean 74 years, controls N � 30, 6M, 24F, age range 37-83 THE HEALING OF DIABETIC PLANTAR FOOT ULCERS OF GREATERyears, mean 63.1 years). THAN TWO-MONTH DURATION WITH GRAFTSKIN IN A

RANDOMIZED PROSPECTIVE STUDY.Microvascular measurements were made while exerting a constantstrain rate (3mm/8seconds) on skin in the distal third of legs, and M. Sabolinski1 and V. Falanga2.

1Organogenesis Inc., Canton, MA; 2Department of Dermatololgy andduring relaxation. These measurements were done on subjects (CVIgroup N � 44, 19 M, 25F, age range 32-81 years, mean 54.3 years, VLU Skin Surgery, Boston University School of Medicine; Roger William’s

Medical Center, Providence, RI.group N � 21, 7M, 14F, age range 34-83 years, mean 65.3 years, controlsN � 18, age range 61-90 years). Subjects were resting supine during

Diabetic foot ulcers of longer duration have been shown to be morethese tests for which the ipsilateral forearm was used as control. Alltests were done with prior informed consent and ethical approval from recalcitrant to healing. These non-healing wounds are more likely to

result in increased morbidity to the patient (e.g. osteomyelitis andthe Local Ethics Committee for this Hospital.amputation). We have previously demonstrated that graftskinsignificantly accelerates the healing of recalcitrant venous leg ulcers.At the most distal site on legs, the frank ulcer group had significantly

stiffer skin compared to both CVI and elderly controls (p � .003 and We, therefore, investigated graftskin, a living, bi-layered skin substitute,on diabetic foot ulcers of greater than two-month duration in ap � .03 respectively Mann-Whitney test). Patients with frank venous

ulcers as well as those with CVI had increased stiffness (p � .006, p randomized, prospective, multi-center clinical trial. One hundredeighty-three consecutive patients were treated who had wounds of� .038 Mann-Whitney test). The increased stiffness is attributed to

changes in the structure and composition of cutaneous tissues subject greater than two-month duration. Patients were randomized to thegraftskin (n � 100) or control groups (n � 83) and were evaluatedto ulcers.over 12 weeks for complete wound closure. Both groups were treatedwith aggressive debridement, off-loading by wheelchair or crutches,During extension, a decrease in blood flow was noted in all groups.

Analysis of the change during relaxation revealed an attenuated and saline dressing changes. By 12 weeks, 55% (55/100) in the graftskingroup attained complete wound closure compared to 36% (30/83) inrecovery in the frank ulcer group compared with other groups (p �

.034, Mann Whitney test). This finding is similar to the impaired the control group, p � 0.0077. We conclude that in a randomized,prospective trial, graftskin heals significantly more patients thanhyperemic response reported on diabetics with skin microangiopathy.control treatment in diabetic foot ulcers of greater than two-monthduration, 55% versus 36%. Use of a living bi-layered skin substitute inThe attenuated recovery in microvascular flow as well the increased

stiffness measured in the frank ulcer group suggest that unrelieved conjunction with debridement, off-loading, and saline dressing changesshould be considered when treating patients with recalcitrantedema leads to tissue stiffness, affects tissue perfusion, and impedes

oxygen diffusion. neuropathic diabetic foot ulcers.

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 149

43. and Allevyn scars were considered to be comparable by the blindedobserver.REPIFERMIN (KGF-2) IS ABLE TO REVERSE DELAYED HEALING

IN PATIENTS WITH HIGH BACTERIAL BURDEN AT PRESENTATION.Split thickness skin graft donor sites dressed with Acticoat healed

Vincent Falanga*, Daniel Odenheimer**, and Partha Bagchi**. Boston more slowly and had more initial scarring with no superiority inUniversity, Providence RI* and Human Genome Sciences, Rockville, bacterial control compared to donor sites dressed with Allevyn. SinceMD.** both dressings create a moist wound healing environment, we cannot

attribute the difference in healing to wet vs. dry environmental effects,The effects of bacterial burden and healing rates on wound closure as might be the case under dressings such as Jelonet, Scarlet Red, orwere evaluated from a 15 center, 94 patient study of the safety and xeroform gauze. We can only conclude that the Acticoat dressingefficacy of repifermin (KGF-2, keratinocyte growth factor-2) in patients inhibited re-epithelialization, through an unidentified mechanism, andwith venous ulcers (VU). Patients were eligible for enrollment in this that our results do not support the use of Acticoat as a skin graft donor12-week study if they had VU 3-30 cm2 in size and 3-36 months in site dressing.duration. Bacterial burden was evaluated by tissue biopsy. Patientswith a bacterial colony count ‘‘ 106/gram of tissue were excluded, but 45.could be enrolled if after initial wound management, had a subsequent IDENTIFICATION OF NOVEL GENES INDUCED BY TRANSFORMINGcolony count < 106. The rate of wound healing was prospectively GROWTH FACTOR-� USING SUBTRACTIVE HYBRIDIZATION.calculated based on the modified Gilman equation. RESULTS: Complete

SD Lee, DR Yager, SI Ward, IK Cohen, and JH Haynes.wound closure was observed for 40% (29/72) of patients with an initialDepartment of Surgery, Medical College of Virginia/Virginiacolony count of < 106 compared with 9% (2/22) of enrolled patientsCommonwealth University, Richmond, VA.with an initial colony count ‘‘106 (p < 0.01). This effect was independent

of wound size and wound duration. For the latter 22 patients, at leastThe transforming growth factor-� is a multifunctional regulator of cell-75% healing was achieved by 62% (8/13) and 11% (1/9) of repifermingrowth, differentiation, and extracellular matrix formation. It isand placebo treated patients, respectively (p � 0.03). Thus,chemotactic for fibroblasts, smooth muscle cells, and macrophages,presentation with a high bacterial count was associated with a failureinduces angiogenesis and modulates expression of collagen andto heal even when measures were taken to lower the counts. However,collagenase. It also has implications in wound contraction. Recently,repifermin appeared to ameliorate this situation. The rate of healingit has been shown to contract fibrin collagen matrix gels in vitro andwithin the first 4 weeks of treatment was predictive of complete woundfetal tissues in vivo in response to the sustained delivery of the variousclosure. An increase in the rate of healing was observed for theisoforms of TGF-� that would otherwise expand. In this study, arepifermin versus placebo treated patients. CONCLUSIONS: Bacterialsubtractive hybridization technique was used to identify the selectiveburden at presentation and the rate of wound healing during the firstpattern of TGF-� isoform induced gene expression. Human fetal4 weeks of treatment were predictors of complete wound healing. Thefibroblast-populated collagen lattices were exposed to TGF-�1 or -�3data indicate that repifermin may reverse the association betweenfor 24 hours and the degree of contraction determined. Up to 80%increased bacterial burden at presentation and delayed wound healing.contraction was observed with TGF-�1 or �3 treated collagen latticescompared to 40% contraction by untreated fibroblasts. Cells were

44. harvested and mRNA isolated. Suppression PCR was used to generateA PROSPECTIVE RANDOMIZED MATCHED PAIR EVALUATION OF subtracted cDNA libraries. Differential screening was performed toA SILVER COATED DRESSING (Acticoat) ON HUMAN SKIN GRAFT further select for cloned cDNAs that were induced by TGF-�1. In aDONOR SITES. first pass screen, twenty three candidate recombinants were selected

and subjected to DNA sequencing. 22/23 of the sequences sharedMarilyn Innes, Rob Cartotto, Joel Fish, Manuel Gomez. Ross Tilley significant similarity with sequences found in the GeneBank or EMBLBurn Unit, Sunnybrook and Womens College Health Sciences Center, databases. Of particular note, one of the recombinant cDNAs wasUniversity of Toronto, Canada. identified as BIGH3, a known TGF-� induced gene product. Quantitative

ribonuclease protection assays were used to further demonstrate thatThe purpose of this study was to compare skin graft donor site healing candidate recombinant cDNAs were induced by TGF-�1. This studyusing Acticoat, against Allevyn,a hydrocellular material which is our validates the suppression PCR subtractive hybridization technique andstandard donor site dressing. The moist healing environment and will be instrumental in identifying candidate genes that are critical insustained silver release provided by Acticoat suggest that it might be mechanisms of tissue contraction in vivo.a useful donor site dressing.

46.Identical matched side by side split thickness donor site wound pairs THE MORPHOGEN WNT-4 IS DOWNREGULATED DURING THEwere each dressed with Acticoat and Allevyn. A single observer EARLY STAGES OF SKIN WOUND HEALING.assessed healing of the wounds from day 6 onwards, and definedhealing as > 90% re-epithelialization. Also, 3 independent observers Robert D. Galiano, Tamara N. Elias, Mary Armour, Oren Z. Lerman,

Jamie Levine and Geoffrey C. Gurtner.rated the extent of re-epithelialization by viewing standardized digitalimages of the wounds that had been obtained on days 6, 8, 10, and 12. Institute of Reconstructive Plastic Surgery, NYU Medical Center, New

York, NY.Donor sites were swabbed for bacterial culture on days 3, 6, and 9.Donor site scarring was rated by a blinded observer at 1, 2, and 3

The Wnt family of morphogens is involved in processes of architecturalmonths using the Vancouver Scar Scale. Results are presented as themean �/� SD, and statistical analysis was performed with either a 2 formation such as body axis determination, organ maturation and skin

development; Wnt-4 in particular is involved in epithelial-mesenchymaltailed paired student’s t test, or a chi square test, where appropriate,with significance ascribed to a p value < 0.05. interactions. We hypothesized that wnt-4 would likewise play a role in

tissue repair, and determined its expression during skin wound healing.16 paired sites in 15 patients were studied.Donor sites dressed with

A partial cDNA encoding the rat Wnt-4 gene was cloned and sequenced,Allevyn were > 90% re-epithelialized at 9.1 �/� 1.6 days while donorsites dressed with Acticoat required 14.5 �/� 6.7 days to achieve 90% and the rat GAPDH probe was purchased. Biotin-labeled riboprobes

were synthesized. For the animal experiments, 21 Sprague-Dawley ratsre-epithelialization (p � 0.004). The mean difference in healing timewas 5.4 �/� 6.7 days. All 16 (100%) of the Allevyn sites were healed were wounded using 6-mm dermal punches down to the level of the

panniculus carnosus. The wounds were harvested at 12 hrs, 1 day, 3by the day of discharge compared to only 5 (28%) of the Acticoat sites(p < 0.001). Based on the evaluation of the standardized digital images, days, 7 days, 14 days and 21 days by excising the wound along with

a 1 mm margin of surrounding skin. RNA was isolated, quantified andAllevyn sites had significantly greater estimated re-epithelializationthan the Acticoat sites at 6, 8, 10, and 12 days. There were no differences utilized in RNAse protection assays. 50 micrograms of each sample of

RNA was hybridized to 2 ng of the wnt-4 and GAPDH probes overnightin the incidence of positive bacterial cultures from either of the sites.Although Acticoat donor site scars were significantly worse at 1 and at 42 degrees, and separated on a 5% polyacrylamide gel. The RNA

was then transferred to a nitrocellulose membrane, crosslinked and2 months, this difference resolved by 3 months, at which time Acticoat

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001150 Meeting Schedule and Abstracts

the hybridized probes were detected with a non-radioactive detection coefficient calculated. Wounds were harvested on the 2nd to 5th and12th postoperative days(d)and stained for collagen, myofibroblasts,kit. The bands corresponding to Wnt-4 and GAPDH were then scanned

and quantified with image-analysis software. and angiotensin receptors. Blood vessel number and area was assessedat 250x magnification within a 25cm2 grid. Results were subjected to

Wnt-4 expression is highest in unwounded skin. As early as 12 hours a t-test.post-injury there is a marked downregulation ( > 50%) in Wnt-4 message;this downregulation persists through the first 7 days postwounding. Wound contraction was significantly slower at all time points in the

dosed animals (contraction coefficients d2: c � 0.07 vs. L � 0.03, d3By day 14 the message is approximately 80% that found in unwoundedskin, and has normalized to the levels found in unwounded skin by � 0.157 vs. 0.02, d4 � 0.15 vs. 0.02, d5 � 0.07 vs. 0.03, d12 � 0.11

vs. 0.07, all time points p£0.05). Blood vessel area (c � 8.2mm2 vs. Lday 21. GAPDH expression did not vary during the course of woundhealing. � 0.7mm2) and number (c � 50 vs. L � 10) was reduced in the d12

dosed animals (p < 0.05). There was a reduction in collagen depositionThis is the first report showing a change in expression of a Wnt in the dosed tissue and alterations in orientation and morphology

observed in the myofibroblast population of treatment groups.morphogen during tissue repair, and it implies a role for morphogensduring the reestablishment of normal dermal structure and scar

Angiotensin converting enzyme inhibitors appear to have specific doseformation in postnatal wound healing. Furthermore, the expression ofbaseline levels of wnt-4 mRNA in unwounded skin suggests a role for dependant effect on angiogenesis, collagen formation and wound

contraction. This suggests that Angiotensin II has an important role tothis morphogen in the maintenance of normal skin architecture. Wnt-4 likely plays a role in directing the epithelial-mesenchymal interactions play in the early development of granulation tissue and wound healing

which has not been previously recognised.that occur during both skin repair and development.

47.49.FIBRIN SEALANT (TISSEEL)TM IS AN IDEAL VEHICLE FOR

HSP47 EXPRESSION IS UP-REGULATED DURING SKIN WOUNDFIBROBLASTS DELIVERY: IN VITRO AND IN VIVO STUDIES.HEALING IN A PORCINE MODE.

Steve Cox, Jon Mogford*, Helena Yoder*, Thomas Mustoe* and NabilTawil*. Wound Healing Research Laboratory, Northwestern University, Jian Fei Wang*, Merle E. Olson*, J. Barry Wright*Y and David A. Hart*.

McCaig Center for Joint Injury and Arthritis Research, Department ofChicago, IL. Wound Mgt. Group, Baxter Healthcare Corp, Duarte, CA.Microbiology and Infectious Disease, Faculty of Medicine, Universityof Calgary, Alberta, CANADA, T2N 4N1, Westaim Biomedical Corp., FtFibrin sealant products, such as Tisseel�, are commonly used in

hemostasis and tissue sealing applications, but in the last several years Saskatchewan, AB.much attention has been given to fibrin sealant products for theirwound healing potential. In this study, we examined Tisseel�as a HSP47 is up-regulated in cells and tissues following heat shock and

during stress responses. Although significant advances have been madepossible vehicle for normal human dermal fibroblast delivery in chronicwound healing therapy. To assess the effectiveness of fibrin sealant as toward elucidating the role of HSP47 as a molecular chaperone in

protein folding and translocation within cells, the specific role thata potential cell delivery vehicle, several parameters, such as cellsurvival/proliferation and migration, were examined. Normal human this molecule plays during wound healing is less clearly defined. HSP47

expression during the process of wound healing was assessed in a welldermal fibroblasts from middle age donors (40-50 years) were harvestedand suspended in the fibrinogen component of the fibrin sealant at established porcine model. Normal skin samples and samples taken

at various time points post-wounding were assessed for HSP47 by250,000cells/ml, placed in a 2.5ml syringe and attached to theDuploject� delivery device along with the thrombin component, also semiquantitative RT-PCR, western-blot, and immunohistochemistry.

The results demonstrated that mRNA levels for HSP47 werein a 2.5ml syringe. Clots were then formed in a 100mm-culture plate(3-4 clots/plate) using a fixed volume and a defined area and allowed significantly elevated during skin wound healing and highly correlated

with HSP47 protein levels. The increases in expression for HSP47to solidify for 1 hour before adding enough fibroblast growth mediumto completely submerge the clots. Proliferation and migration of during wound healing also correlated with collagen I and collagen III

expression. Immunohistochemistry studies using an anti-HSP47fibroblasts were then monitored by staining with Calcein and analyzingsubsequent digital images of the clots for an increase in cell number antibody revealed that HSP47 was constitutively expressed in normal

skin at low levels, and the levels in tissues were highly correlated withand migration distance of the cells. Our data shows that fibroblastssurvive and proliferate well within the three-dimensional fibrin matrix. mRNA and protein levels at various time points in the healing process.

Furthermore, based on immunolocalization studies of samples takenAdditionally, fibroblasts were able to migrate from the clot continuallyfor the duration of the assays (18 days) with cells retaining good at various times post-injury, HSP47 protein was exclusively expressed

by fibroblasts rather than inflammatory cells, keratinocytes, ormorphology and growth characteristics. We have also successfully usedTisseel to add mouse fibroblasts to chronic wounds in mice. We endothelial cells in this model. These observations suggest that

expression of HSP47 during porcine wound healing is highly regulatedconclude that fibrin sealant is an excellent delivery vehicle forfibroblasts and likely applicable to most wound situations, especially and the molecule likely plays a significant role as a molecular chaperone

for collagens during extracellular matrix repair. Whether HSP47 alsochronic wounds.serves other functions in skin wound fibroblasts remains to be resolvedand is currently being further evaluated.48.

THE EFFECTS OF LISINOPRIL DURING EARLY WOUND HEALINGIN THE RAT.

50.S.W. McKirdy*, B. Chew, I. Naylor, D.T. Sharpe. Plastic Surgery and ASSESSMENT OF ACUTE TENSILE STRENGTH IN WOUNDSBurns Research Unit, University of Bradford, BD7 1DP, United CLOSED WITH FIBRIN TISSUE ADHESIVE.Kingdom.

James W. Doyle, Erin Wirth, Yaye Lo, and David Tuthill. American RedCross, Holland Laboratory, Rockville, MD.In order to assess the development of granulation tissue, fibrosis and

wound contraction in the early phase of wound healing we examinedFibrin sealants (FS), or fibrin tissue adhesives, are natural andthe effects of the Angiotensin Converting Enzyme inhibitor Lisinopril.biodegradable products that have hemostatic and tissue adhesiveproperties. Current fibrin sealant products are formulated primarily5 groups of rats (n � 4) were dosed daily by gavage with 10mg/kg of

Lisinopril/methylcellulose suspension(l), 5 control groups(c)(n � 4) for hemostatic applications and may require modifications for optimaltissue adhesion. This study has examined the acute tensile strength ofwere dosed with methylcellulose. 144mm2 full thickness squares of

skin were excised from the left flank. Wound areas were measured fibrin sealant with various concentrations of fibrinogen and using aquantitative in vivo technique.daily (n � 10/wound/day), digitised, and the wound contraction

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 151Two full-thickness 2 cm incisional wounds were made on the 52.

ONTOGENETIC TRANSITION IN THE FETAL WOUNDdorsolateral surfaces of the thorax of male Sprague-Dawley rats (~350g). FS for use as a fibrin tissue adhesive was prepared from solutions EXTRACELLULAR MATRIX CORRELATES WITH SCAR FORMATION.of human topical fibrinogen complex (TFC) and thrombin that were S. Beanes, C. Dang, C. Soo, P. Benheim, M. Hedrick, and H.P. Lorenz.mixed during application to the wound in a two-syringe dispenser. A Department of Surgery, UCLA, Los Angeles, CA.total of 100 microliters of FS was applied to each wound and the edgesof the wound were held together for 5 minutes. The ultimate (bursting) INTRODUCTION: Fetal rat wounds transition from scarless repair topressure was measured using a BTC-2000 (SRLI, Nashville) at 1 or 2 repair with scar between gestational days 16 and 18 (Term � 21.5hours following wound closure. days). Expression differences in extracellular matrix (ECM) molecules

are likely to be critical in this transition from scarless repair. LysylInitial studies examined the kinetics of acute tensile strength in wounds oxidase (LOX), an ECM enzyme that crosslinks collagen and elastin,closed with FS. For these studies, a formulation containing a low has been implicated in fibrotic disease, cellular differentiation, andconcentration of thrombin (3 units/ml) was used because it was growth. Fibromodulin, an ECM proteoglycan, has potential anti-fibroticbelieved that fibrin polymerization, and hence wound strength, would regulatory roles in both collagen assembly and TGF-� bioactivity.require a longer time to develop than with formulations using higher Decorin is an ECM proteoglycan that regulates TGF-� bioactivity andthrombin concentrations. Surprisingly, it was found that the ultimate assists in collagen fibrillogenesis. We hypothesize that LOX,pressure decreased 34% (P � 0.0265) between 1 and 2 hours following fibromodulin, and decorin will be differentially expressed during thewound closure. fetal wound transition period.

Subsequent studies examined the acute tensile strength of nine FS METHODS: Excisional wounds were created on fetal rats gestationalformulations at 1 hour after wound closure. These formulations ages 16 (E16) and 18 (E18). Wounds were harvested at 24 and 72 hourscontained TFC at 30, 60, or 120 mg/ml and thrombin at 3, 30, or 300 (N � 12 wounds/time point). Non-wounded fetal skin (E17 to E21)units/ml. FS formulations composed of 120 mg/ml TFC and 3 or 30 was used as control. Reduced cycle specific primer reverseunit/ml thrombin had a significantly greater ultimate pressure than did transcriptase polymerase chain reaction was performed to determineformulations containing 30 mg/ml TFC or 300 units/ml thrombin. LOX, fibromodulin, and decorin expression. Unpaired two-tailed

student’s t-test was employed (p < 0.05 significant).The conclusion of this study is that FS with high thrombinconcentrations (i.e., hemostatic formulations) or low fibrinogen RESULTS: As a function of fetal skin development, fibromodulinconcentrations (e.g., ‘‘homemade’’ from patient cryoprecipitate) have expression significantly decreased (p < 0.0003), while both LOX andsuboptimal acute tensile strength and thus may have limited utility as decorin expression significantly increased (p < 0.0006 and p < 0.01,fibrin tissue adhesives. The results of this study also suggest that the respectively). In fetal wounds, fibromodulin expression wasacute tensile strength of wounds closed with FS rapidly decreases, significantly increased at 24hrs in E16 wounds (p < 0.00004), whileperhaps by proteolytic degradation of fibrin-tissue crosslinks. decorin expression was significantly decreased both at 24 and 72hrs

(p < 0.05). LOX expression was significantly decreased in E16 wounds51. at 72hrs (p < 0.02). Increased fibromodulin expression in scarless

KELOID AND NORMAL FIBROBLASTS IN MONOLAYER AND RAFT wounds was confirmed with immunohistochemistry.CULTURES: THE TGF�1 EFFECT ON APOPTOSIS DEPENDS ON THELEVEL OF STRESS. CONCLUSIONS: There are distinct differences in ECM molecule

expression between pre-transitional and post-transitional fetal wounds.C. C. Chipev and M. Simon. Living Skin Bank and Department of OralPre-transitional wounds exhibit down regulation of pro-fibroticBiology and Pathology, University Hospital and Health Sciences Center,molecules, such as decorin and LOX, and up-regulation of anti-fibroticSUNY at Stony Brook, Stony Brook, NY.molecules, such as fibromodulin. These data suggest that differentialLOX, decorin, and fibromodulin regulation in pre- and post-transitionalKeloids are excessive accumulations of collagen, which occur infetal wounds may contribute to scarless repair. Potential clinicalnonglabrous skin and extend beyond original wound margins. Whenapplications include anti-LOX, anti-decorin, and pro-fibromodulincompared to fibroblasts derived from nonwounded glabrous ortherapeutic strategies to affect scarless wound healing.nonglabrous skin, keloid fibroblasts in monolayer cultures express

more a-smooth muscle actin (a-SMA) and do not as readily initiate53.apoptosis in response to serum withdrawal.

CELL PHENOTYPE AND MICROENVIRONMENT INHIBIT HEALINGOF CHRONIC VENOUS LEG ULCERS.In order to determine whether the level of a-SMA determines the entry

into apoptosis, these two parameters were measured by two- Michael Stacey, Michael Woosey and Hilary Wallace. UniversityDepartment of Surgery, Fremantle Hospital, Fremantle, Westerndimensional FACS for cultures grown under a variety of conditions

mimicking different stages of wound healing. Fibroblasts were Australia.incubated in the presence or absence of 10ng/ml TGF�1 while underdifferent levels of stress developed by growth on tissue culture plastic Impaired healing of chronic venous ulcers could be due to impaired

function of the resident cells in the wound or to a lack of stimulation(high stress), collagen I coated plastic (low stress), or within attachedor detached collagen gels. The extent of apoptosis correlated positively of these cells from the wound environment. The aim of this study was

to compare the rates of proliferation of fibroblasts obtained fromwith increased cell and matrix relaxation. Thus, more apoptosis wasseen in cultures grown on collagen-coated plastic than on non-coated chronic venous leg ulcers with fibroblasts from normal skin and

foreskin under normal culture conditions and in the presence of acuteplastic, and more apoptosis was seen in relaxed collagen gels than inattached (stressed) gels. Growth with TGF�1 increased a-SMA levels or chronic wound fluid.but was found to be either anti-apoptotic or pro-apoptotic dependingon the resulting state of the matrix (stressed vs. relaxed). When the Fibroblasts were obtained from the edge of 3 chronic venous leg ulcers,

leg skin of 3 age matched patients and foreskin from 3 infants. Acutelevel of a-SMA was increased and stress was maintained apoptosis wasinhibited. However, in keloid fibroblasts that had a high initial wound fluid (AWF) was pooled from mastectomy patients as was the

chronic wound fluid (CWF) from 4 venous leg ulcer patients. Earlyconcentration of a-SMA, further treatment with TGF�1 caused gelrelaxation and increased apoptosis. passage (P3-4) fibroblasts from all cell lines were then incubated in

either DMEM or DMEM with 10% fetal calf serum (FCS) in the presenceOur data show that myofibroblasts undergo apoptosis. The dependence of increasing concentrations (up to 25%) of AWF or CWF. At 48 hours

cell numbers were determined using a crystal violet assay.of apoptosis on TGF�1 is a complex function of serum and stress. Inorder to explain the in vivo occurrence of keloids we must assume

Foreskin fibroblasts proliferated at a greater rate than either ulcer orthat there is sufficient time for excess collagen deposition prior tomatrix contraction and the ensuing loss of fibroblasts due to apoptosis. normal age matched cells (ANOVA, p < 0.05). Normal age matched

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001152 Meeting Schedule and Abstracts

cells proliferated at a greater rate than ulcer cells in DMEM/FCS but flow in the rabbit ear wound, showing that wounds at days 2 and 8were stimulated, and small increases in blood flow persisted up to 14not in DMEM alone. AWF and CWF at low conventrations ( < 3.125%)

stimulated similar proliferation in all cell lines.At higher days. Studies with the same materials in diabetic wound models confirmthese effects. This partially purified biological material appears toconcentrations( > 3.125%) of AWF, proliferation was significantly less

for ulcer cells (P < 0.05). In contrast to AWF, CWF at higher contain many key constituents for stimulating wound repair.concentrations did not increase proliferation rates in all 3 cell types.

56.

BACTERIAL DEGRADATION OF GROWTH FACTORS.Impaired wound healing in chronic venous leg ulcers is in part due tothe decreased ability of resident fibroblasts to proliferate and to the F.Ko, T.E.Wright, W.Payne, X.Wang, M.C.Robson. Institute for Tissuereduced ability of the wound environment to stimulate proliferation. Regeneration, Repair, and Rehabilitation, Bay Pines, FL.

54. Application of exogenous growth factors has been shown to overcomePOTENTIAL ROLE OF CHEMOKINES IN WOUND CONTRACTION the inhibition to contraction caused by bacteria. However, it requiredAND CLOSURE. very large doses of the growth factors. For this reason, it is

recommended to decrease the bacterial burden when using growthJo Ellen Feugate and M. Martins-Green.factors in clinical trials. It has been suggested that bacterial enzymesDepartment of Cell Biology and Neurosciences, University of Californiasuch as proteases or the production of MMPs from interaction ofRiverside, CA.bacteria and tissue may cause degradation of the growth factors.Methods: 5 million organisms of each; Psuedomonas aeruginosa, E.Chemokines are small cytokines that are primarily known for theircoli, Staphlococcus aureus or Streptococcus faecalis were mixed withrole in inflammation. However, they are increasingly being implicated500pg/ml of TGF-�2. The mixtures were incubated at 36 degrees C inin other functions such as in tumor development and in the formationa 5% carbon dioxide incubator. TGF-�2 at 500pg/ml without bacteriaof the granulation tissue during wound healing. These molecules areserved as a control. Result: P. aeruginosa and E. coli degraded theexpressed highly by fibroblasts, cells that play key roles in theTGF-�2 to 264.85 � /-14.50pg/ml and 263.01 � /-10.02pg/ml respectivelydevelopment of the healing tissue. They proliferate, migrate into thein 3 hours. No further significant degradation occurred at 24 hours.wound site and produce extracellular matrix molecules that areThe gram positive organisms (S. aureus and S. faecalis) degraded TGF-important in the healing process. In addition, during wound healing,�2 to 329.47 � /-15.82pg/ml and 339.75 � /-49.84pg/ml respectively insome fibroblasts differentiate into myofibroblasts, cells that are3 hours. Again, no further significant degradation occurred at 24 hours.important for proper wound contraction. They express theAdding fibroblasts to the mixture further degraded the TGF-�2 by allmyofibroblast marker a-smooth muscle actin (a-SMA), and acquire thefour bacterial species to levels ranging from 230.82 � /-23.34pg/ml andability to contract and close the wound. We have previously shown195.59 � /-14.65pg/ml. Control level of TGF-�2 remained near the initialthat cCAF, a chicken CXC chemokine with high homology to several500pg/ml level at 3 hours and 24 hours (457.58 � /-13.43pg/ml andhuman chemokines including IL-8, MGSA/groa and �TB, is highly421.50 � /-25.76pg/ml). Conclusion: TGF-B2 is degraded by 42.2% in 3expressed during granulation tissue formation, the phase of woundhours by gram negative rods and degraded 27.7% by gram positivehealing when fibroblasts differentiate into myofibroblasts. Usingcocci. When bacteria are in the presence of soft tissue cells such ascultures of chicken embryo fibroblasts (which have properties similarfibroblasts, they further degrade the growth factors. These data supportto those of wound fibroblasts) under conditions that mimic the woundthe control of bacterial burden prior to application of exogenous growthenvironment, we also showed that cCAF increases the levels of a-SMAfactors.and stimulates fibroblasts to strongly contract collagen gels. These

effects of cCAF on myofibroblast differentiation can also be57.accomplished by a 16 amino acid peptide from the N-terminus of the

FETAL AND ADULT SKIN FIBROBLASTS HAVE DIFFERENTIALmolecule. Here, we show that cCAF and the amino terminus haveRESPONSES TO TGF�.similar effects in vivo. Treatment of excision wounds with exogenous

cCAF or the N-terminal peptide accelerates wound closure. Sections Veronique Moulin, Francois Auger, Maureen O’Connor-McCourt, Luciethrough the wounded tissue show that at early stages after injury Germain. LOEX, Quebec; Biotechnol. Res. Instit., Montreal, Canada.treated wounds have granulation tissue of a more mature appearance,with many elongated, myofibroblast-like cells. In summary, cCAF Fetal skin wounds heal without contracture and scar formation. Threestimulates myofibroblast differentiation resulting in faster closure of adult skin fibroblast lines (AF) and 3 fetal skin fibroblast lines (FF)wounds in vivo and this stimulation is localized to the N-terminus of were cultured for 7 days without or with TGF�1, �2, and �3. Then,the molecule. We are currently investigating the mechanism of the N- fibroblast populated-collagen gels were produced and cultured withterminal peptide activity and also the effects of human chemokines or without TGF�. Capacity of the cells to contract was evaluated withwith similar amino termini on myofibroblast differentiation. gel diameter measurements. Then, gel was put in collagenase H solution

and cells were counted. The percentage of a-SM actin-immunolabeledcells was evaluated and flow cytometry analysis was performed with55.anti-human a1, a2, a3 or �1 integrin. Without previous treatment, initialBONE-DERIVED GROWTH FACTOR ACCELERATES WOUNDamount of contraction was quite similar between FF and AF. Then,HEALING.the rate of FF-mediated gel contraction was lower, and diameter gels*†Jeffrey M. Davidson, *Jeffrey S. Whitsitt, *Rebecca Parker, �Johnbecame higher than that of the AF. At day 14, the number of AF in gelRanieri, �Rama Akella. Department of Pathology*. Vanderbilt Universitywas increased in opposition to FF number which was constant. WithSchool of Medicine and VA Medical Center†, Nashville, TN; SulzerTGF�, FF showed an inhibition of their contractile capacity whereasBiologics �, Austin, TX.AF further contracted gels. FF number did not show any changewhereas a slight growth of AF was noted. The expression of a-SM actinBiological sources of vulnerary agents have been sidestepped in theand a3 and �1 integrin subunits was increased when TGF� was addedrush to develop recombinant DNA strategies. We have carried out ain the medium of AF whereas FF showed a decrease of a1, a2 and �1set of validation procedures with a novel mixture of growth factorsintegrin expression. These results indicate that intrinsic differences(GFM) derived from bovine bone. The GFM was applied to linearexist between FF and AF. These variations represent potentiallyincisions in the rat at the time of surgery or both the time of surgeryimportant mechanisms used by FF to regulate their response, and likelyand 3 days later. With a single dose, tensile strength of incisionscontribute to the resultant differences noted in the quality of woundincreased 50-80%, and with double dosing wound strength was elevatedrepair.50-100% at both 7 and 10 days after surgery. The polyvinyl sponge

implant model in the rat confirmed that GFM was able to increase thecollagen content of granulation tissue, particularly at 7 days afterimplantation. There was a relative doubling of this component. In therabbit ear ulcer model, application of 50�g of GFM resulted in largeincreases in wound granulation tissue area and a doubled rate of woundclosure. Laser Doppler perfusion imaging was used to estimate blood

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 153

Conclusions: These results suggest a combined role for rhPDGF-BB58.

A HIGH RATIO OF TGF�3 TO TGF�1 EXPRESSION IN WOUNDS IS and TGF-� during collagen contraction by adult human dermalfibroblasts. These mediators may act through a convergent pathwayASSOCIATED WITH SCARLESS REPAIR.at Rho-GTPase. PI-3’ kinase may mediate rhPDGF-BB but not TGF-�

C. Dang, S. Beanes, C. Soo, M. Hedrick, H.P. Lorenz. Department of effects during collagen contraction.Surgery, UCLA, Los Angeles, CA.

60.Purpose: The regulatory events coordinating the transition fromOXYGEN GROWTH ENVIRONMENT-DEPENDENT ACTIVATION OFscarless healing at 16.5d gestation to healing with scar formation atHUMAN DERMAL FIBROBLAST P42/P44 MAPK BY HYPERBARIC18.5d gestation are unknown in the rat fetus (Term � 21.5d). BecauseOXYGEN.TGF� is implicated in both skin development and scar formation, we

hypothesize that differential expression of the TGFbeta isoforms (- Alexandrina S. Saulis, John D. Davidson, Thomas A. Mustoe, Jon E.�1, -�2, -�3) and their receptors (T�R-I, -II, -III) may participate in the Mogford. Northwestern University, Chicago, IL.regulation of the transition in repair outcome.

Hyperbaric oxygen (HBO) treatment is known to promote the healingMethods: Full-thickness, 2mm excisional wounds were created on the of problematic human dermal wounds. In an ischemic rabbit ear model,dorsal surface of fetal rats at gestational days 16.5 (E16) and 18.5 (E18). we demonstrated HBO and PDGF-b act in synergy to accelerate woundWounds were harvested at 24 and 72hrs (N � 12 wounds/time point). healing and that this synergy also upregulates PDGF receptor b (PDGF-Non-wounded fetal skin was used as control. Reduced-cycle specific Rb). However, the underlying cellular mechanisms of these effects areprimer reverse transcriptase polymerase chain reaction was performed unknown. We hypothesize that HBO treatment directly modulatesto determine TGF� isoform and receptor expression. Two-tailed intracellular signal transduction pathways similar to peptide growthstudent’s t-test was employed (p < 0.05 considered significant). factors. To test this hypothesis, we measured p42/p44 MAP kinase

activation by HBO treatment of human dermal fibroblasts. p42/p44 areResults: TGF�1 expression was increased 77% in E18 wounds at 24hrs cytosolic kinases known to modulate growth factor-dependent cellular(p < 0.02), while its expression was unchanged from control in E16 responses such as proliferation, migration and gene expression.wounds. TGF�2 was increased 32% at 24hrs in E16 wounds (p < 0.02), Primary human dermal fibroblasts (n � 6), grown in three oxygenand decreased 72% in E18 wounds at 72hrs (p < 0.01). TGF�3 expression environments: 1%, 2.5% and 5% (control) O2 for 3 weeks, were thenwas upregulated 100% at 24hrs in E16 wounds (p < 0.04), and decreased treated with HBO (2.4 ATA, 2h), normobaric (NBO; 100% O2, 1 ATA)46% at 24hrs in E18 wounds (p < 0.01). At 24hrs, T�R-II expression or control O2 pressure (5% O2, 2.4 ATA) for 4 days then lysed 30was increased 38% and 111% for E16 and E18 wounds, respectively (p minutes after last treatment for western blot analysis of p42/p44< 0.05 for both). Expression of T�R-I and -III did not differ from baseline activation. HBO activated p42/p44 to a greater extent in cells grownfor either E16 or E18 wounds. at 5% O2 (p < 0.05) while cells grown in 1% O2 responded strongest

to NBO. Cells grown at 2.5% O2 displayed equal activation of the kinaseConclusion: In scarless wounds, the expression of TGF�2 and �3 was to HBO and NBO. Additionally, HBO-treated 5% growth cells alsoupregulated, while TGF�1 unchanged. In fetal wounds that healed with showed a 3-fold increase in erk1/2 activation by PDGF-B (2 ng/ml)scar, TGF�1 expression was increased, while TGF�2 and �3 expression compared to control groups, similar to our in vivo observation of HBO-was decreased. These data demonstrate differential expression of PDGF synergism in the ischemic rabbit ear model to controls. WeTGF� isoforms in scar and scarless wounds. In scarless wounds, the conclude that HBO significantly increases p42/p44 activity dependentTGF�3/TGF�1 ratio was high, while the ratio was low in scar-forming on the prevailing oxygen growth environment. Furthermore, exogenouswounds, which suggests that changes in TGF� isoform expression PDGF-B synergizes with HBO to increase p42/p44 activity as comparedprofile may affect repair outcome. to control groups. These data support the concept that HBO activates

fibroblasts using a similar signal transduction cascade as traditionalpeptide growth factors.59.

rhPDGF-BB AND TGF-� INDUCE COLLAGEN CONTRACTIONTHROUGH CONVERGENT SIGNALING. 61.

PDGF-STIMULATED MIGRATION OF HUMAN DERMALYuan-Ping Han, Michael Hughes, Warren L. Garner FIBROBLASTS IN 3-D COLLAGEN GELS IS REDUCED WITH AGEDivision of Plastic and Reconstructive Surgery, University of Southern AND HYPOXIA.California School of Medicine; Los Angeles, California 90033, USA.

Jon E. Mogford and Thomas A. Mustoe.Wound Healing Research Laboratory, Div. of Plastic Surgery,Introduction: Cutaneous wound undergoes contraction although a

molecular mechanism has not been fully clarified. A role for PDGF Northwestern University Medical School, Chicago, IL.and TGF-� during in vitro and in situ collagen contraction (deleted)has been individually established but the mechanism for these effects An important component of cutaneous wound healing involves the

migration of dermal fibroblasts from surrounding tissue into the injuredis unclear. Of the potential pathways, phosphatidylinositol-3’ kinaseand Rho-GTPase have been suggested to mediate growth factor induced area or hemostatic plug. This requires movement cellular movement

through a 3-D extracellular matrix in response to injury cues such ascytoskeletal remodeling.cytokines and matrix fragments. We established a 3-D collagen gelchemotaxis assay to test the migratory response of human dermalMethods: Adult dermal fibroblasts were embedded in collagen, treated

with PDGF-BB (0 - 100 ng/ml) and TGF-� (0 - 10 ng/ml) and lattice fibroblasts (HDFs) to PDGF under a normal culture oxygenenvironment (20% O2) and under hypoxia (1% O2). We additionallycontraction measured. PI-3’ kinase activity was quantified. Using

inhibitors, wortmannin for PI-3’ kinase, and Clostridium botulinum tested the migratory differences HDFs isolated from healthy donorsbetween aged ( > 60 years of age) and young ( < 30 years of age).Toxin B for Rho-GTPase, we determined the roles of these signaling

molecules during collagen contraction. Based on previous work in vivo and in other in vitro systems, wehypothesized migration would be negatively impacted by age and

Results: Individually, rhPDGF-BB at 25 ng/ml and TGF-� at 2.5 ng/ml ischemia. HDFs (2x105 cells; n � 6 each group) were embedded in acollagen gel placed in a cell culture insert with a porous filter base.induced maximal collagen contraction. At lower concentrations,

combined rhPDGF-BB (2.5 ng/ml) and TGF-� (1 ng/ml) treatment Media alone or containing PDGF-B (5 ng/ml) was placed in theunderlying well so that the porous insert membrane was submerged.resulted in similar, maximal collagen contraction. The additive effects

suggested a possible common pathway. Clostridium botulinum Toxin After 2 days, cells adherent to the membrane stained, the stain waseluted and read at 590 nm. Young cells migrated out of the gel to aB at 0.4 ng/ml blocked both rhPDGF-BB and TGF-� induced

contraction, which suggests that Rho-GTPase may be the convergent greater extent under normoxia and hypoxia with media alone asstimulus (p < 0.05 for both conditions). This may reflect a greater basalpoint for rhPDGF-BB and TGF-� induced collagen contraction.

rhPDGF-BB but not TGF-� induced collagen contraction was blocked migratory activity of the young cells. Compared to controls, additionof PDGF to the media enhanced the difference between the aged andby wortmannin, which suggests a role of PI-3’ kinase in this activity.

Indeed, PI-3’ kinase was stimulated by rhPDGF-BB but not TGF-�. young cells under normoxia (from 22% to 60% difference) and hypoxia

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001154 Meeting Schedule and Abstracts

(16% to 57%). Unexpectedly, aged HDFs displayed a significantly had a Km of 0.34 �M, a Kcat of 8.44/min and a Vm of 127 nM/min.Similarly, both ribozymes targeting CTGF had good kinetic parameters:increased migration to PDGF under hypoxia vs. normoxia (p < 0.05).

The data support the concept that young cells possess a greater C2041 had a Km of 1.76 �M, a Kcat of 30.3/min and a Vm of 454 nM/min; C2472 had a Km of 7.80 �M, a Kcat of 66.7/min and a Vm of 1000migratory capability in response to wound-associated stimuli.nM/min. Only one of the two ribozymes targeting TGF-betaIIR cleavedthe target RNA: Tr1555 had a Km of 11.9 �M, a Kcat of 333.33/min and62.

a Vm of 5000 nM/min.DIFFERENTIAL EXPRESSION OF CONNECTIVE TISSUE GROWTHFACTOR BY TRANSFORMING GROWTH FACTOR-� IN FIBROBLAST

Synthetic hammerhead ribozymes can specifically cleave the syntheticMONOLAYER VS. FIBROBLAST-POPULATED COLLAGEN LATTICE.RNA substrate for TGF-beta1, TGF-beta type II receptor and CTGF.

SD Lee, SI Ward , JH Haynes, and DR Yager. Department of Surgery, CR � none and support from NIH EY 05587Medical College of Virginia/Virginia Commonwealth University,Richmond, VA.

64.

Connective tissue growth factor (CTGF) is a cysteine-rich peptidewhich is a mitogenic and chemotactic for fibroblasts. It acts as adownstream mediator of TGF-� and has been implicated in woundhealing and fibrotic disorders. So far, expression of TGF-�1 is the onlyknown inducer of CTGF and it has been shown to stimulate time anddose dependent increases in CTGF.

Recently, TGF-� has been shown to contract fibrin collagen matrixgels in vitro and fetal tissues in vivo in response to the sustaineddelivery of the various isoforms of TGF-� that would otherwise expand.With TGF-�’s implication in wound contraction, the role of CTGF incontraction was explored. In this study, the expression of CTGF inhuman fetal dermal fibroblasts was measured using ribonucleaseprotection assays. The two study models included fibroblast monolayerand fibroblast-populated collagen lattice (FPCL). Interestingly, bothTGF-�1 and �3 induced CTGF expression in the FPCL model. However,only TGF-�1 was shown to induce CTGF in dermal fibroblastmonolayer. There appears to be an inherent environmental factor thataffects CTGF expression. In addition, if FPCL is an accuraterepresentative of an in vivo model, this study suggests CTGF may playa role in wound contraction. Both FPCL contraction and CTGFexpression were comparable with TGF-�1 and �3. As suggested byprevious studies, there is low level TGF-�3 expression in fetal tissueswhich could translate into low grade CTGF expression and thus in anabsence of contraction. Exogenous TGF-�1 and �3, which in turn willincrease expression of CTGF, induce contraction of fetal open dermalwounds. This study may be the stepping stone for better understandingthe mechanism of wound contraction as we explore downstream fromTGF-�.

63.

KINETIC ANALYSIS OF HAMMERHEAD RIBOZYMES AGAINST TGF-beta1, TGF-beta TYPE II RECEPTOR AND CTGF mRNAs.

R Yuan, TD Blalock, JC Varela, MH Goldstein, AS Lewin, GS Schultz.Institute for Wound Research and Dept Ophthalmology and DeptMolecular Genetics and Microbiology, Univ. of Florida, Gainesville, FL. 65.

DIFFERENTIAL EXPRESSION OF VASCULAR ENDOTHELIALCytokines play key roles in the wound healing process. The GROWTH FACTOR IN FETAL WOUNDS.transforming growth factor (TGF-beta) signaling system has beenwidely shown to promote tissue repair by stimulating fibroblast S. Beanes, C. Dang, C. Soo, P. Benheim, M. Hedrick, and H.P. Lorenz.

Department of Surgery, UCLA, Los Angeles, CA.proliferation and collagen synthesis. Recently, connective tissue growthfactor (CTGF) has been shown to mediate the scarring effects of the

Introduction: Vascular Endothelial Growth Factor (VEGF) is a dimericTGF-beta system.heparin-binding glycoprotein that is a potent endothelial cell-specificmitogen with increased expression during adult cutaneous woundOne method for selectively decreasing the activity of TGF-beta and

CTGF systems is to use hammerhead ribozymes that target the mRNAs healing. VEGF activity is mediated by two receptors with tyrosinekinase activity, FLK-1 and FLT-1, which are expressed mainly in vascularfor TGF-beta1, TGF-beta type II receptor (TGF-betaIIR) and CTGF. The

advantage of ribozymes is that they can repeatedly cut target RNA like endothelial cells. The regulatory events coordinating the transitionfrom scarless healing at 16.5d gestation to healing with scar formationa protein enzyme. Two hammerhead ribozyme cleavage sites were

identified in each of the mRNAs for human TGF-beta1, TGF-betaIIR at 18.5d gestation are unknown in the rat fetus (Term � 21.5d). BecauseVEGF is implicated in skin development and adult wound healing, weand CTGF. Corresponding 33mer RNA hammerhead ribozymes were

chemically synthesized and designated as, T673, T1526 for TGF-beta1; hypothesize that differential expression of VEGF and its receptors mayregulate the transition from scarless wound healing to healing withTr865, Tr1555 for TGF-betaIIR, and C2041, C2472 for CTGF. Target

12mer mRNAs were synthesized containing the cleavage site and scar formation.labeled with gamma 32P-ATP. Kinetic parameters (Vm, Km, Kcat) weremeasured by performing time course reactions and multi-turnover Methods: Excisional wounds were created on fetal rats gestational

ages 16 days (E16) and 18 days (E18) (term � 21 days). Wounds werereactions.harvested at 24 and 72 hours (N � 48 wounds). Non-wounded fetalskin (E17, E19, and E21) was used as control. Reduced cycle, specificSingle and multi-turnover studies showed that both ribozymes targeting

TGF-beta1 had excellent kinetic parameters. Specifically, T673 had a primer, reverse transcriptase polymerase chain reaction was performedto determine the expression of VEGF and its receptors, FLK-1 and FLT-Km of 7.54 �M, a Kcat of 83.3/min and a Vm of 1250 nM/min. T1526

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 155

By day 7, wounds of the treatment groups were significantly smaller1. Unpaired two-tailed student’s t-test was employed (p < 0.05 wasconsidered significant). than control (control � 108.0 � /-6.6%, HBO plus LED � 72.5 � /-

5.9%, and LED alone � 80.5 � /-6.6%). Both VEGF and FGF-2Results: During normal skin development, VEGF expression increased expression peaked at 4 days in control (VEGF � 1.97 � /-0.31ng/mg

protein, FGF-2 � 60.2 � /-7.4ng/mg protein) and LED treated wounds2.4-fold (p < 0.001) during the transition from scarless healing to scarformation (E17 to E19). In scarless wounds (E16), VEGF expression (VEGF � 1.64 � /-0.23ng/mg protein, FGF-2 � 80.8 � /-28.8ng/mg

protein). Treatment with HBO alone significantly blunted the initialincreased 2.8-fold (P < 0.02) at 72hrs. No increased expression occurredin the scar wounds (E18). FLK-1 and FLT-1 expression increased during response to hypoxia (VEGF � 0.44 � /-0.40ng/mg protein, FGF-2 �

13.4 � /-11.6ng/mg protein at 4 days), with a peak at 7 days (VEGFnormal skin development, however, each was down-regulated inscarless (E16) and scar (E18) wounds. � 1.03 � /-0.08ng/mg protein, FGF-2 � 52.5 � /-4.9ng/mg protein).

The combination of the two modalities resulted in early stimulationConclusion: VEGF and its receptors, FLK-1 and FLT-1, show increased of these angiogenic factors (FGF-2 > VEGF) which was sustained

throughout the healing phase.expression during normal skin development. VEGF expression quicklyelevates in scarless wounds compared to scar wounds, which likely

These results demonstrate that the effect on angiogenic factors in thiscontributes to the rapid scarless fetal repair rate.model is augmented when HBO and LED are used in combination.This data supports our hypothesis that combining LED treatments with66.HBO will overcome the early inhibitory effect of hyperoxia onADENOVIRAL-MEDIATED ANGIOPOIETIN-1 OVEREXPRESSIONangiogenesis. Ongoing studies are analyzing the effects on matrixACCELERATES RE-EPITHELIALIZATION IN IMPAIRED WOUNDmetalloproteinases and the relationship between the angiogenic factorsHEALING.and neovascularization.

F. Lim, J. Karmacharya, A. Gordon, B. Martin, B. Edelman, A. Radu,68.R. Kirschner, K. Satymoorthy, M. Herlyn, and T. M. Crombleholme.

AN INVESTIGATION INTO THE IMPAIRED WOUND HEALINGChildren’s Institute for Surgical Science at The Children’s Hospital ofPHENOTYPE OF FIBROBLASTS FROM GENETICALLY DIABETICPhiladelphia, PA. The Wistar Institute, University of Pennsylvania,MICE.Philadelphia, PA.

Oren Z. Lerman, Robert D. Galiano, Tamara N. Elias, Ernest Chiu andAngiopoietin-1 (Ang1) is a vasculogenic factor which mediates its effect Geoffrey C. Gurtner.through the endothelial cell-specific Tie2 receptor tyrosine kinase Institute of Reconstructive Plastic Surgery, NYU Medical Center, Newknown to modulate neovascularization. The effect of Ang1 in wound York, NY.healing is unknown. We hypothesize that adenoviral mediated Ang1overexpression in excisional wounds in genetically diabetic (db/db) The db/db diabetic mouse is a recognized model of impaired woundmice may enhance local angiogenesis and accelerate wound healing. healing. We investigated the cellular defects that might account forTwo 10mm excisional wounds were created in the dorsum of db/db this impairment. We studied dermal fibroblast migration and productionmice and were treated by direct injection of either 1x10e8 pfu (50 of the angiogenic mediator VEGF, two cellular functions important inmicroliters) of Ad-Ang1 or Ad-LacZ, and similar volume of PBS for tissue repair.control. Animals were sacrificed at 3, 7, and 14 days post woundingfor histological evaluation by three independent individuals. Histologic Fibroblasts were harvested from C57BL/K mice and C57BL/K-db/dbsections were scored for epidermal reaction, vascular response, mice via collagenase digestion and grown in DMEM/10%FBS. For theconnective tissue formation, and inflammatory response. In contrast gold salt phagokinetic migration assay the cells were seeded at 5000to Ad-LacZ treated wounds which did not heal until day 14, Ad-Ang1 cells per well onto 8-well plates coated with collagen and gold salts.treated wounds were completely re-epithelialized with minimal signs The collagen concentrations varied from 0ug/ml to 20 ug/ml. After 20of inflammation and marked vascularization by 7 days. We concluded hours the migration paths of the cells through the gold salts werethat adenoviral mediated overexpression of Ang1 accelerates re- digitally photographed under inverted darkfield microscopy. Theepithelialization and overall healing of impaired wounds in db/db mice. migration tracks were then quantified with image-analysis software.Thus, Ang1 may play a previously unrecognized role in wound healing, For analysis of VEGF production, cells were plated onto 10 ug/mland adenoviral-mediated gene transfer of Ang1 may prove effective in collagen-coated culture dishes at a concentration of 20,000 cells/cm2.accelerating tissue repair in wound healing. After 20 hours the media was concentrated, its protein content

quantified and was then analyzed by ELISA for VEGF concentration.67.

THE EFFECT OF LIGHT-EMITTING DIODE THERAPY AND Both diabetic and normal fibroblasts migrated and produced VEGF.HYPERBARIC OXYGEN ON ANGIOGENESIS. Diabetic fibroblasts, however, show a severe impairment in both of

these processes. Normal and diabetic fibroblasts migrated optimallyLJ Gould, M Kane, G Chen, HT Whelan. Medical College of Wisconsin, at a collagen concentration of 10 ug/mL. At this concentration, however,Milwaukee, WI. diabetic fibroblasts exhibited markedly less migration than normal

fibroblasts (4-fold less, p < 0.05). Furthermore, diabetic fibroblastsThe true etiology of chronic wounds remains elusive. One common secreted less than half the total VEGF produced by normal fibroblastsdenominator, whether the wounds are due to pressure, diabetes, or on both plastic (60 pg/mL vs 124 pg/mL, p < 0.05) and type I collagen-arterial insufficiency is localized tissue ischemia. This study was coated plates (31 pg/mL vs 78 pg/mL, p < 0.05).designed to test the hypothesis that two physical modalities, light-emitting diode therapy (LED) and hyperbaric oxygen (HBO), promote These results highlight the impaired physiology of diabetic fibroblastsangiogenesis in hypoxic wounds. at the cellular level. Cellular migration and angiogenesis are key

functions in wound healing. The identification of defective cellularA modification of the ischemic wound model originally described by mechanisms impairing these events would allow for the rational designSchwartz was utilized. Full thickness excisional wounds were created of therapeutics directly targeting these processes. Further investigationwithin a bipedicled dorsal skin flap in rats. Insertion of a silicone sheet into the cellular physiology of diabetic fibroblasts is critical for thebeneath the skin flap prevented re-adherence and reperfusion of the elucidation of the molecular pathology underlying impaired woundflap from below. There were four experimental groups: control (n � healing in diabetic patients.15), LED treatment at 880nm (n � 15), HBO treatment (n � 9) andLED at 880nm plus HBO (n � 15). LED and HBO treatments weregiven daily for 14 days, with the LED delivered at a fluence of 4J/cm2and HBO at 2.4 atm for 90 minutes. Wound surface area tracing andexcision of the wound beds was performed on days 4, 7, and 14. Theconcentration of VEGF and FGF-2 in wound extracts was quantifiedby ELISA.

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001156 Meeting Schedule and Abstracts

69. 71.

ANALYSIS OF THE MATRIX METALLOPROTEINASE PROFILE INTHE LACK OF TSP1 PREDOMINATES IN DETERMINING THECOURSE OF WOUND HEALING IN DOUBLE TSP1/TSP2-NULL MICE. DIFFERENT CHRONIC WOUNDS AFTER SURGICAL

DEBRIDEMENT.Azin Agah, Themis R. Kyriakides, Jack Lawler, and Paul Bornstein.Department of Biochemistry, University of Washington, Seattle, WA C. Riordan, L. Bennett, K. Zahir and L. Nanney. Department of Plasticand Department of Pathology, Beth Israel Deaconess Medical Center Surgery and Cell Biology, Vanderbilt Medical Center, Nashville, TN.and Harvard Medical School, Boston, MA.

Aggressive surgical debridement is known to promote wound healingThe matricellular inhibitors of angiogenesis, thrombospondin (TSP)1 in chronic wounds. To study the pathogenesis behind this clinicaland 2 are structurally similar and have been reported to interact with observation wound fluid was collected from paraplegic decubitusa number of the same cell surface receptors. However, TSP1- and TSP2- ulcers (n � 16), normally innervated chronic wounds (n � 11), acutenull mice display distinct phenotypes. We have developed double TSP1/ dermal wounds (n � 7), and breast reductions (n � 6). Fluids fromTSP2-null mice to elucidate their biological roles in the highly the chronic and acute cutaneous wounds were collected at 24, 48, 72orchestrated process of wound healing. Full thickness 6mm-diameter hours and 1 week after surgical debridement and placement of a woundwounds were made on the backs of TSP1-, TSP2- and double TSP1/ vacuum device. Fluids from the breast reductions were collected fromTSP2-null mice, and wounds were harvested 7 and 14 days post-injury. surgical drains at 24 and 48 hours post surgery. Total human matrixGross examination of wounds revealed that, similar to TSP1-null mice metalloproteinase-13 (MMP-13), MMP-8 and MMP-2 in the samples wasbut in contrast to the accelerated healing observed in TSP2-null mice, determined using the Biotrak sandwich ELISA system (Amersham,double TSP1/TSP2-null animals demonstrated delayed healing, as Little Chalfont, Buckinghamshire, UK).indicated by signs of inflammation and the persistence of scars at day14. However, granulation tissue from double TSP1/TSP2-null wounds At 24 hours post debridement both MMP-8 and MMP-13 were minimallyresembled that of TSP2-null wounds in having abnormally organized elevated in the chronic wound types compared with the acute dermalcollagen fibers. Wound neovascularization was quantitated with the wounds. MMP-13 was not detected in the fluid from the breastaid of imaging software (Metamorph) following immunolocation of the reductions at 24 or 48 hours. At 72 hours, MMP- 13 and MMP-8 wereendothelial cell marker, PECAM-1. Neovascularization of granulation elevated six-fold and two-fold respectively in the chronic wound typestissue in double TSP1/TSP2-null mice was similar to that observed in compared with the acute dermal wounds. Of note, MMP-13 was presentTSP1-null mice but significantly lower than in TSP2-null mice [day7: in the denervated chronic wounds at a significantly higher level (p <5.2 � 1.8 vs. 7.5 � 1.9 (TSP1) and 14.6 � 6.2 (TSP2); day 14: 5.4 � 0.05) than present in the normally innervated chronic wounds. MMP-3.4 vs. 4.7 � 2.5 (TSP1) and 13.5 � 3.8 (TSP2)]. No significant 2 levels (both total and active) were elevated in all the chronic woundsdifferences in the levels of TGF-beta and MMP2 were observed in the at both 24h and 72h.wound sites of TSP1/TSP2-null mice in comparison with the findingsin single knockout animals. These results suggest that wound healing These results demonstrate that the process of surgical debridementis predominantly influenced by the absence of TSP1 in mice lacking appears to temporarily change the MMP-13 and MMP-8 profile of aboth TSP1 and TSP2. chronic wound to that of an acute wound. This phenomenon may

explain the improvement in chronic wound status seen post70. debridement. This alteration is not sustained and higher collagenase

CHARACTERIZATION OF THE MOUSE MMP-13 PROMOTER IN levels prevail in the chronic wounds at 72 hours and persist at 1-weekVITRO AND VISUALIZATION OF ACTIVITY IN LIVING, TRANSGENIC post debridement. This study also shows that the predominatelyMICE. fibroblast derived MMP-13 is present in significantly higher quantities

in all chronic wound fluids compared to normal acute dermal fluids.Nan Jun Wu†, Jin-Hua Liu†, Jeffrey S. Whitsitt†, Jeffrey M. Davidson†§.Of significance, the higher levels of MMP-13 in the denervated†Department of Pathology, Vanderbilt University School of Medicinecompared to normally innervated chronic wounds may highlight subtleand §Veterans Affairs Medical Center, Nashville, TN.wound healing abnomalities among these subtypes of wounds.

Collagenase-3 (MMP-13) is the matrix metalloproteinase primarily72.responsible for the turnover of the interstitial collagens type I, II and

RATIOS OF ACTIVE MATRIX METALLOPROTEINASE-9/TISSUEIII in the mouse. In the human, its expression is more limited, but itsINHIBITOR OF MATRIX METALLOPROTEINASE-1 IN WOUNDtranscription is regulated by growth factors, mediators of inflammation,FLUIDS ARE INVERSELY CORRELATED WITH HEALING OFoncogenes, phorbol ester tumor promoters and carcinogens. ByDECUBITIS ULCERS.cleaving interstitial collagen, MMP-13 likely plays a role in wound

repair. A mouse embryonic stem cell genomic DNA library was screened Glenn P. Ladwig, Martin C. Robson, Ran Liu, M. Ann Kuhn,David F.using a 540bp fragment of the known mouse MMP-13 promoter as a Muir, Gregory S. Schultz; from the Institute for Wound Research,probe. A 6.5kb fragment which contained a 1.8kb fragment upstream Department of Obstetrics and Gynecology, University of Florida,of the translation initiation site was identified. Several constructs Gainesville, FL, the Institute for Tissue Regeneration, Repair andharboring MMP-13 promoter truncations and a luciferase reporter gene Rehabilitation, Bay Pines VAMC, Bay Pines, FL, and the Departmentwere developed based on this 1.8kb promoter region. Assay of promoter of Neuroscience, University of Florida, Gainesville, FL.activity after transient and stable transfection of normal human skinfibroblasts, HT1080 human fibrosarcoma cells, and mouse normal skin Previous studies examining wound fluids collected from acute healingfibroblasts delineated several functional regions within the promoter. wound and chronic non-healing wounds found elevated levels ofExpression of reporter genes by HT1080 cells and mouse skin inflammatory cytokines (TNFa and IL1b), elevated levels of proteasesfibroblasts was induced by TPA and inhibited by TGF-�2. To study the (MMPs, NE, and plasminogen activator) and low levels of growth factorregulatory mechanism of MMP-13 expression in vivo, several transgenic activity in chronic nonhealing wounds. This led to the hypothesis thatmouse lines have been generated in which the mouse MMP-13 promoter, chronic inflammation produced elevated levels of proteases thatligated to the luciferase reporter gene, has been stably integrated into destroyed growth factors, receptors and ECM proteins that are essentialthe mouse genome. Northern blot showed that luciferase and MMP- for healing.13 mRNAs were expressed by the skin fibroblasts from transgenicmice. Luciferase activity was visualized in real time during the course There are no reports comparing the rates of healing of chronic pressureof wound repair. Results showed that promoter activity increased into ulcers with the levels of MMPs and TIMPs in fluids. This study examinedthe remodeling phase, beginning at the wound margin and proceeding a series of 56 patients with pressure ulcers that were treated withto the center of wound. This system will be useful for the study of conventional therapy or with exogenous cytokine therapies (bFGF,MMP modulation during the course of wound repair. Supported by GM-CSF, or sequential GM-CSF followed by bFGF). Wound fluids andNIH grant AG06528, the Office of Naval Research, and the Department biopsies were collected and wound volume measurements were takenof Veterans Affairs. at days 0 (pre-treatment), 10 and 36. Levels of pro and active MMP-9

and MMP-2 were measured by quantitative gelatin zymography andlevels of TIMP-1 and TIMP-2 were measured by ELISAs. The identity

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 157of the MMPs were confirmed through immunoblot. It was found that tissue development in contaminated full-thickness porcine wounds.

The current study evaluated the impact of different dressing treatmentsthe minimum amount of MMP-2 and MMP-9 that could be detected bythe gelatin zymograms under identical incubation conditions was not on the MMP activity in recovered wound fluid. Full-thickness wounds

were created on the dorsum of domestic pigs and contaminated withequal. Specifically, the limit of detection (sensitivity) for the pro andactive forms of MMP-9 was approximately 8-fold lower than the limit a bacterial suspension. The wounds were dressed with pieces of

Acticoat� silver coated high density polyethylene, Acticoat� Burnof detection for the pro and active forms of MMP-2, producing animportant and potentially misleading visual effect when samples of dressing, water-moistened gauze, and silver nitrate-moistened gauze

followed by an occlusive covering. On a daily basis, the dressings werewould fluids and biopsies are analyzed.replaced and the used dressings were used to recover wound fluid.The MMP activity in the wound fluid was determined by enzymeThe zymogram and ELISA data from wound fluids and biopsy

homogenates were categorized into cytokine treatment groups and zymography as well as fluorogenic enzyme activity assays. The resultsof repeated experiments yielded very similar data. The MMP activityclinical response groups based on ulcer volume over time. Multivariate

analysis of variance revealed that active MMP-9 levels in wound fluids level in the Acticoat� silver-treated wounds was higher than in thecontrols on Day 1. Zymography data suggested that this was due towere significantly (p < 0.05) higher at day-0 in those patients that

healed slower (poor responders) than those patients that healed more elevated MMP-9 (active) activity. Throughout the duration of theexperiments (to Day 7) the control and silver nitrate-treated woundsrapidly (good responders). Likewise, TIMP-1 levels were significantly

(p < 0.05) higher in good responders at day-0 than poor responders. demonstrated increasing MMP activity. However, over the same periodof time, the Acticoat� silver-treated wounds demonstrated a virtuallyIn the ratio of total (pro � active) mean MMP-9 to TIMP-1, significant

differences were found at day-0 between good responders, poor constant level of MMP activity. Interestingly, the modulated MMPactivity in the Acticoat� silver-treated wounds corresponded to similarresponders and intermediate responders. Levels of pro MMP-2 and in

wound fluids at day-0 between good responders and poor responders wounds in previous experiments that healed approximately 7 daysearlier than the control wounds. These data further support the conceptapproached statistical significance (p � 0.077). While there was no

statistical relationship found between MMP or TIMP level and cytokine that modulation of MMP activity may correlate with an enhancementof wound healing and that nanocrystalline silver is an effectivetreatment group, these data provide strong additional support for the

concept that high levels of protease activity and low levels of protease modulator of MMP activity in experimental wounds.inhibitors are detrimental to healing.

75.

MATRIX METALLOPROTENASES AND TISSUE INHIBITOR OF73.

METALLOPROTEINASE ARE DIFFERENTIALLY EXPRESSEDTARGETED EXPRESSION OF STROMELYSIN-1 IN KERATINOCYTESBETWEEN ACUTE MURINE EXCISIONAL AND LASER WOUNDS.OF TRANSGENIC MICE REGULATES EARLY STAGES OF WOUND

HEALING. Bradley K. Draper,*† Mari K. Davidson,* and Lillian B. Nanney #†*Departments of Medicine (Dermatology), †Cell Biology and #PlasticLisa J. McCawley, Jane Wright and Lynn M. Matrisian. Department ofSurgery, Vanderbilt University School of Medicine, Nashville, TN, U.S.A.Cancer Biology, Vanderbilt University, Nashville, TN.

Matrix metalloproteinases (MMPs) and their physiological inhibitors,This study focuses on the role of the matrix metalloproteinase (MMP)the tissue inhibitors of metalloproteinases (TIMPs) play significantStromelysin-1 during reepithelialization. MMPs are a family ofroles in normal cutaneous wound healing. Using in situ hybridization,extracellular matrix degrading proteinases implicated in variety ofwe previously demonstrated both spatial and temporal differences innormal and pathological cellular processes including wound healing.the expression patterns of MMP-1, -2, -9 and TIMP-1 among partial-Stromelysin-1 is rapidly expressed following wounding by boththickness excisional, laser and burn wounds in the pig. In this study,fibroblasts and keratinocytes. Mice null for Stromelysin-1 have delayedwe investigated whether MMP and TIMP-1 mRNA is differentiallywound closure. To address the function of Stromelysin-1 duringexpressed between acute, full-thickness, laser-created skin wounds andreepithelialization in vivo, we have generated transgenic mice withexcisional skin wounds. While normal, ten-week old, male Balb/c micetargeted expression of Stromelysin-1 in keratinocytes using the bovinewere under anesthesia, six full-thickness wounds (6mm in diameter,Keratin 5 promotor (K5-Str-1). Constructs containing both the full4 mm apart) were created on the dorsal surface by either excision oflength wildtype rat stromelysin cDNA and an constitutively activethe skin or by using a CO2 laser. Animals were sacrificed at days 1, 3,construct which contains an amino acid substitution at position 935, 7, 10 and 13 respectively, and their wounds were harvested by(pro-val) were utilized. Full thickness 3mm dorsal wounds werecomplete excision and processed either for RNA isolation or foradministered in wildtype and K5-Str-1 transgenic mice. Time to closurehistologic evaluation and immunohistochemistry. Non-wounded dorsalwas moderately enhanced in the K5-Str-1 transgenic mice as comparedskin served as controls. Northern hybridization for MMP-1, 3, 10 andto wildtype controls, with the differences in rate of closure most notableTIMP-1 mRNA demonstrated higher expression on days 1, 3, and 5 inat early time points. Analysis of tissue sections by histology usingthe laser-created wounds as compared to the excisional wounds. TheGomori’s Trichrome stain and immunohistochemically using antibodiesvariability in expression kinetics between the individual MMPs, andagainst keratin 5 and keratin 6 revealed that the migratory front ofTIMP-1 suggest that laser-induced transcriptional enhancement is notkeratinocytes in K5-Str-1 transgenic mice was altered at early stagesmerely a generalized up-regulation of transcription. Histologicalof wound closure as compared to wildtype controls. We conclude thatevaluation revealed a one to two day delay in the resurfacing of laserStromelysin-1 expression in keratinocytes influences early eventswounds as compared to excisional wounds. Immunohistochemicalregulating the migratory front of keratinocytes duringanalysis of day 5 and 7 excisional and laser wounds showed substantialreepithelialization.MMP-3 protein within the epidermal compartment and a diffusedistribution in the neo-dermis. No significant differences in MMP-374.immunoreactivity were noted between the two different wound types.MODULATION OF MATRIX METALLOPROTEINASE ACTIVITY BYIn summary, our findings demonstrate that MMPs and TIMP-1 mRNANANOCRYSTALLINE SILVER DRESSINGS IN EXPERIMENTALexpression is differentially higher on days 1-5 post injury in fullWOUNDS.thickness laser-created wounds than in comparable same day excisional

J. Barry Wright, Kan Lam, and Robert E. Burrell. wounds. The mechanisms of laser induced up-regulation of MMP-1, -Westaim Biomedical Corp., Fort Saskatchewan, AB. 3, -10 and TIMP-1 remain as yet undefined.

Chronic ulcers of various etiologies have been characterized as havingexcessive levels of matrix metalloproteinase (MMP) activity. A highlevel of MMP activity correlates well with delayed healing in venousstasis ulcers. As a result, recent research efforts have focused onvarious methods of inhibiting MMP activity in wounds so that theirimpact on wound healing may be evaluated. Previous studies utilizingnanocrystalline silver dressings (Acticoat� Burn Dressing) haveindicated that the use of the dressing enhances the rate of granulation

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001158 Meeting Schedule and Abstracts

76. in both groups and no significant change was noticed for serum CREATfor both groups of mice. The younger group of mice had a strongerANTIMICROBIAL PEPTIDES ARE EFFECTORS OF WOUND REPAIR.LDH and CK peak response than older mice at 3 hours after burn.

V.K. Pestonjamasp and R.L. Gallo. Interestingly, compared to other serum protein changes, CK responseUniversity of California, San Diego, La Jolla California. was extremely transient and it already returned to the basal level at

day 1 after burn.Antimicrobial peptides play a vital role in innate immunity by providinga chemical barrier to microbial colonization. In addition, they are These findings that serum response after burn injury is differentlyversatile effector molecules that influence neutrophil function, attract regulated dependent upon age may explain the different clinicalT-lymphocytes and induce synthesis of extracellular matrix outcomes between age groups after burn injury. Further understandingcomponents. The cathelicidin class of antimicrobial peptides (human of the underlying mechanisms of changes in serum proteins will helpLL-37 and murine CRAMP (Cathelin Related Antimicrobial Peptide)) elucidate the signaling pathway of systemic response after burn injury.are present in granulocytes but are not detected in normal epidermis.However, upon injury cathelicidins are heavily expressed in the skin

78.at the wound site. Since wounding presents a high likelihood ofTHE LOCALIZATION OF HUMAN BETA DEFENSIN-2 IN BURNEDsubsequent infections, synthesis of antimicrobial peptides would be ofSKIN BY FLUORESCENCE IMAGING.paramount importance to the host in restraining the spread or the

severity of infection. Cathelicidins in skin may also act directly on host Stephen M. Milner, Christopher E. Smith, Brian J. Poindexter,cells to change their behavior, thus aiding events important to wound Maximilian Buja, Roger J. Bick. Regional Burn Center, Memorialhealing. To examine the mechanisms that lead to cathelicidin Medical Center, Institute for Plastic and Reconstructive Surgery,expression, we have stimulated HaCaT cells with known mediators of Southern Illinois University, Springfield, Illinois, and Department ofinflammation. Using quantitative RT-PCR by LightCycler, we find that Pathology, UT Houston.the tumor promoter phorbol myristate acetate (PMA), humanrecombinant interferon-� (IFN-�) and bacterial lipopolysaccharide Introduction: As sepsis is a serious complication of burn injury, the(LPS) modulate LL-37 expression. In addition, we have detected LL- role of antimicrobial peptides has become an important factor for37 protein synthesis in response to IFN-� by dot blot analysis. The 5’ consideration. Our previous work has detailed the reduced expressionflanking region of the murine cathelicidin CRAMP gene also shows of human b defensin-2 (HBD-2) in burned tissue. This cysteine-richbinding sites for a number of transcription factors known to be peptide has potent antibacterial activity against gram-positive, gram-important in the inflammatory cascade. To further study the regulation negative, and fungal organisms. Our aim was to determine byof cathelicidin expression, truncations of the 5’ flanking region of the deconvolution fluorescence microscopy which cell types in skin weremurine cathelicidin CRAMP gene have been carried out. These able to synthesize HBD-2, whether these cells are destroyed in burnconstructs were cloned into a luciferase reporter vector and the wounds, and to determine the level of the skin in which they appear.luciferase activity has been determined in HaCaT cells. Preliminarydata indicate evolutionarily conserved regions of the CRAMP promoter Methods: Specimens of burned and non-burned skin were sectionedthat regulate gene expression. Significance of cathelicidins in wound and then fixed in paraformaldyhde. Incubation in 10% goat seruminfections is being further studied using transgenic animals challenged reduced non-specific binding. Antibody to HBD-2 was then incubatedwith Streptococcus A, a known serious skin pathogen. CRAMP gene with the specimens, followed by FITC and Texas red tagged secondary‘‘knockout’’ mice show much greater number of streptococci colonizing antibody. Samples were then reacted with fluorescent actin stain andthe wound, show more severe wound infections, and take a longer specimens were visualized with an Applied Precision Delta Visiontime to heal as compared to the wild type animals. In conclusion, we deconvolution microscope. Images were acquired through the samplesshow that cathelicidin expression can be modulated in response to at 0.1 to 0.25 microns section thickness and presented as stackedinflammatory mediators and is an important aspect influencing the section images.wound healing process.

Results: Images of normal skin reveal that HBD-2 is localized in thebasal layer. Smaller amounts are found throughout the epidermal77.

thickness up to the keratinocytes at the skin surface. This contrastsAGE-RELATED CHANGES IN SERUM PROTEINS AFTER BURNwith the appearance of burned skin. Despite the presence of anINJURY IN MICE.epidermal layer in some specimens, there is almost total absence of

Kiho Cho, Lee K. Adamson, and David G. Greenhalgh. HBD-2 with only a few remnants in the basal layer and deeperShriners Hospitals for Children Northern California and Department structures, possibly eccrine glands. Actin in normal skin is fibrillar,of Surgery, University of California at Davis, Sacramento, CA. coursing between cells to produce a supportive, structural assembly.

In burned skin, the actin is somewhat globular and disarranged.Cutaneous burn injury causes skin damage as well as a systemicresponse, called the systemic inflammatory response syndrome. One Conclusions: The imaging of human skin samples utilizing fluroescentlyof our research focuses related to the burn injury is to understand the tagged antibodies illustrates the synthesis of HBD-2 in the basal layermechanism of how the local wound affects the entire organism. We and migration to the skin surface via keratinocytes. It further suggestshypothesize that the maturation of the immune system affects the that epidermal cells in the dermis may also produce this peptide.intensity and timing of the systemic immune reaction as well as serumprotein changes after burn injury. We investigated the effects of burn

79.injury on the serum protein changes in 2 different age groups of mice. THE DETERMINATION OF BURN WOUND DEPTH USING

MAGNETIC RESONANCE IMAGING.Two age groups (7 weeks and 13 weeks) of female BALB/c mice weresubjected to 18 % TBSA full-thickness burn. Blood samples were Christopher E. Smith, George R. Magre, W. Ross Stevens, Stephen M.

Milner.collected at 4 different time points (3 hours, 1 day, 7 days, and 21 days)after burn. There were 6 mice at each time point (3 burns and 3 no Deparments of Surgery and Radiology, Southern Illinois University,

Springfield, Illinois.burn controls). Eight different serum proteins (BUN, CREAT, ALT, AST,ALKP, amylase, LDH, and CK) were analyzed on a Roche COBAS Mira

Introduction: The diagnosis of burn depth has always been a constantinstrument.challenge. The differentiation between indeterminate, superficial, anddeep burns is crucial in subsequent surgical management. This studySerum ALT levels peaked at 3 hours and 1 day after burn for 7 weeks

and 13 weeks old mice, respectively and started returning to the basal examines MRI as a useful method of predicting burn depth.level thereafter. Serum AST levels in both groups of mice weresignificantly up-regulated 3 hours after burn. ALKP was slightly elevated Methods: Nine patients aged 17 to 65 with extremity burns of varying

depth underwent MR imaging of the burned area. The burn depth ofat day 1 and down-regulated at later time points in both groups. Theserum amylase level was slightly decreased in the older group and each wound as determined clinically. Axial images were obtained using

conventional spin echo T1, fast spin echo T2 fat-suppressed, and fastincreased in younger group at 3 hours. Serum BUN peaked at 3 hours

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 159spin echo inversion recovery (FSEIR). Markers were placed on the 81.

NON-CONTACT LASER DOPPLER IN BURN DEPTH ESTIMATION.wounds to identify the burned area on MRI images. Images obtainedwere interpreted by a radiologist blinded to both the history and

C.Riordan, C Perlov, R Corley, M McDonough, J Guy, R Barton, Lappearance of the burns. A full thickness 1 cm diameter core biopsyNanney.was taken from each burn wound imaged and examined histologically.Departments of Plastic Surgery and Cell Biology, Vanderbilt UniversitySchool of Medicine, Nashville, Tennessee.Results: T1-, T2-, and FSEIR MRI images showed clear delineation

between superficial and deep burns. Superficial partial thickness burnsEarly excision and grafting of burns lowers mortality, decreaseswere noted on T2-weighted images to appear with a uniform mildlyinfection rate and scar formation and reduces hospital stay. The mainincreased signal throughout the epidermis and superficial dermis. Thisobstacle to early excision and grafting of the burn wound isindicates edema in these areas. Full thickness burns showed extensionidentification of the deep areas. The purpose of this prospective studyof this increased signal intensity beyond the epidermis and superficialwas to determine the accuracy and utility of a prototypic non-contactdermis into the deep dermis and superficial fat. Furthermore, this signallaser Doppler Imager in the estimation of burn depth. Serial scans ofwas confluent in T2-weighted images indicating greater tissue36 indeterminate burns in 23 patients were performed at 24, 48, anddestruction in the deeper skin layers. Histological sections from each72 hours post burn. Histological samples were obtained on all burnswound confirmed the accuracy of the MRI diagnosis in all 9 cases. Theby either 3mm-punch biopsy or tissue specimen removed at excisionhistology also confirmed the accuracy of clinical diagnosis in eightof the burn. The scanning head of the imager is mounted on a flexiblecases.arm of a mobile stand for ease of positioning. As the beam executesa raster pattern across the burn, the laser Doppler flux value isConclusions: Previous modalities in burn imaging have disadvantages.computed immediately and each value is represented as a colored pixelUltrasound has variability in interpretation, there are narrow windowson a screen. Thus a burn perfusion ‘map’ of the wound is formulated.of accuracy with thermography, and the use of radioisotopes and vitalThe laser Doppler flux value measures tissue perfusion and scalesdyes has potentially dangerous systemic effects. Our results using MRI,linearly with the product of red cell velocity and concentration. Fromwhich is noninvasive and nondestructive, may overcome many of theseour experience a flux value less than 0.5 represented a full thicknesslimitations. Burn depth as determined by MRI appears to correlate wellburn, that between 0.5 and 1.3 a deep dermal burn and values greaterwith both histological and clinical predictions, making it a potentiallythan 1.3 indicated a superficial burn. For outcome analysis, histologicalvaluable tool for evaluation of the burn wound.samples were analyzed for burn depth with vimentinimmunohistochemical staining.

80.

NEAR INFRARED SPECTROSCOPY ASSESSMENT OF BURN DEPTHThe laser Doppler imaging scans showed flux greater than 1.3 in allIN THE IMMEDIATE POST-BURN PERIOD.burns that were confirmed by histology to be superficial dermal in 94%

Joel S. Fish, Lorenzo Leonardi, Michael G. Sowa, Jeri R. Payette, Henry of cases. Burns with flux between .6 and 1.3 were shown to be deepH. Mantsch. Ross Tilley Burn Centre, Sunnybrook & Women’s College dermal on histology in 9 out of 10 burns. Finally, progression in burnHealth Sciences Centre, Toronto, ON, Canada, and Institute for depth over 72 hours was seen in sequential scans of deep dermal burns.Biodiagnostics, National Research Council of Canada, Winnipeg, MB,Canada. This study demonstrates the advantage of using a non-contact laser

Doppler to accurately differentiate deep dermal and superficial partialThe purpose of this study was to demonstrate the potential of the non- thickness burns. It involves minimal discomfort to the patient, givesinvasive near infrared (NIR) point spectroscopy and imaging technique reproducible results and can be performed easily at the bedside.to accurately evaluate the depth of thermal damage in the early post-burn period based on hemodynamic tissue changes, in a porcine model. 82.

ROLE OF NEUTRAL ENDOPEPTIDASE INHIBITION IN DIABETICFive pigs were used in the study, each with four wounds (superficial, WOUND REPAIR.intermediate partial, deep partial, and full thickness burns) created on

ML Spenny, P Muangman, JE Olerud*, and NS Gibran. Departments ofthe dorsal surface. NIR point spectra and images were acquired priorSurgery and *Dermatology, University of Washington, Seattle, WA.to the burns and immediately following the burn at hourly intervals

for 4 hours. NIR spectra provided a measure of tissue hemodynamicsInflammation comprises a crucial initial event for normal wound repair.(balance between oxygen delivery and oxygen utilization) for bothIn response to cutaneous injury, sensory nerves release substance Pinjured and uninjured tissue.(SP), a neuropeptide with proinflammatory effects on endothelial cellsand keratinocytes. Neutral endopeptidase (NEP) a cell surfaceTissue oxygen saturation (StO2) images provide a qualitative visualmetallopeptidase degrades SP to regulate its activity. Chronic non-survey of the burn. Images of the dorsal region involving the uninjuredhealing ulcers from patients with diabetes have increased NEPsites appear bright which indicates normal tissue oxygenation.localization and activity. We hypothesized that NEP activity would beHowever, the injured sites, which display a drop in oxygenationincreased in obese diabetic mutant mice and that NEP inhibition wouldfollowing the thermal insult, appear as dark areas in the post-burnimprove closure kinetics in an excisional wound model. Skin samplesimages. Hemodynamics determined from NIR point spectroscopyfrom diabetic C57BLKS/J-M � / � Leprdb mice (db/db) and non-provides a quantitative measure of thermal damage. An ANOVA analysisdiabetic heterozygous (db/-) littermates were homogenized. NEPshows a significant alteration in StO2 over time (p < 0.05) relative toenzyme activity was determined by DL-thiorphan inhibitablethe uninjured tissue. Although total hemoglobin shows no significantdegradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine. A full-change (p � 0.456) over time, a difference exists comparing burnthickness dorsal excisional wound was created with a 6 mm biopsydepths (p < 0.001). Individually, the hemodynamic parameters cannotpunch on db/db and db/- mice. Wounds were covered with a semi-distinguish the various burn depths. However, a multivariate analysisocclusive dressing and treated daily with normal saline (NS) or thethat considers both parameters over time is capable of differentiatingNEP inhibitor thiorphan (25 �M) for 7 days. Mice were followed untilall four burns. A multivariate pair-wise Hotteling T2 tests indicates thatwound closure when they were euthanized and wounds were harvestedthe burns can be reliable distinguished at an alpha < 0.025.for H&E analysis. NEP enzyme activity in unwounded db/db skin (20.6pmol MNA/hr/ug) significantly exceeded activity in db/- skin (7.9 pmolThe depths of thermal injuries were identified immediately followingMNA/hr/ug; p � 0.02). In db/db mice, thiorphan (p < 0.05) shortenedinjury based on the relative change and distribution of thetime to closure (18.0 d) compared to NS treated wounds (23.5 d). NEPhemodynamic parameters determined from NIR spectroscopy.inhibition did not alter wound closure kinetics in the db/- mice. Therewas no difference in the size of healed wounds between the groupssuggesting that contraction was not affected by thiorphan. Histologicevaluation of healed wounds showed no difference in inflammation orgranulation tissue between db/db treatment groups using a semi-quantitative scoring system with two blinded observers. In unwounded

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001160 Meeting Schedule and Abstracts

skin from diabetic mice, we show increased NEP activity, which may Immunohistochemistry of E15 to E16 fetal wounds showed aproliferation of keratinocytes in the epidermal layer noted by andecrease the availability of substance P and impede early inflammation.

NEP inhibition improved wound closure kinetics without affecting increase in basal keratinocyte staining and proliferating cell nuclearantigen (PCNA) staining surrounding the wound. Furthermore,contraction suggesting that epithelialization may be augmented.

Whereas the healed wounds demonstrated no difference in granulation upregulation of urokinase plasminogen activator (uPA) andplasminogen activator inhibitor-1 (PAI-1), as well as matrixtissue, inflammation may be enhanced in early wounds treated with

an NEP inhibitor. metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2), were seen in the epidermal layer at the site of injury. Thedifferential expression of matrix degrading proteases and the early re-83.epithelialization seen in fetal wounds may contribute to the scarlesscDNA MEMBRANE MICROARRAY GENE ANALYSIS OF RATrepair seen after fetal injury. (Supported by NIH grant 1RO1GM55081CORNEAS FOLLOWING EXCIMER LASER PHOTOREFRACTIVEto T.-L. Tuan.)KERATECTOMY.

JC Varela, MH Goldstein, HV Baker1, GS Schultz. Institute for Wound85.

Research and Depts of Ophthalmology, Molecular Genetics and HYDROGEN PEROXIDE: PRODUCTION AND DESTRUCTION INMicrobiology1, University of Florida, Gainesville, FL. WOUNDS.

Purpose: To analyze the patterns of gene expression of 1176 genes in LM Humphrey, N Puzziferri, TK Hunt. Department of Surgery, Universityof California at San Francisco, San Francisco, CA.rat corneas after PRK and to determine the relationships among them

and the role of specific genes in corneal wound healing. Methods: PRKMolecular oxygen (O2) performs many essential functions in wounds.ablation was performed to a depth of 60 m on 60 corneas of Sprague

Dawley rats. On days 3 and 7 after ablation, 30 corneas were harvested Some is converted to bactericidal oxidants and some acts as a signalto VEGF production. The fraction normally converted is unknown.and pooled into three samples of 10 corneas each, total RNA was

isolated using TriZol and 5 mg of RNA were labeled with 32P ATP Excessive production is potentially harmful. We hypothesize that alarge portion of the O2 that is delivered to wounds is converted tousing RT and gene specific primers provided by Clontech. Three pooled

samples of normal corneas each consisting of 10 corneas were similarly oxidants, but antioxidant defenses provide competent protection evenwhen oxygen delivery is enhanced.processed. Labeled cDNA was hybridized to 9 new array membranes

and the signal intensities measured by digital phosphorimaging. DataWound oxygen tension (pO2) was measured continuously in guineawere analyzed using Atlas Image 1.5 Software (Clontech) and net

hybridization intensity for each gene was normalized using variance- pigs using an implanted electrode before and during breathing of 100%oxygen. Diphenylene iodonium was then added to inhibit NADPHnormalization calculation. The top 50% of genes with the largest

standard deviation (i.e., the largest change in expression) were then oxidase, the initial step in oxidant generation. H2O2 was measured inthe extracellular fluid before and after inhibition of catalase,analyzed using Cluster and Tree View software programs to identify

genes based on their common expression patterns across the three myeloperoxidase and addition of hydrogen peroxide.time points. Results: 591 genes were divided into 8 clusters based onthe similarities of their gene expression patterns. 61 wound healing- Wound pO2 increased about three-fold (80 � /- 20 mmHg to 240 � /-

20 mmHg) after inhibiting NADPH oxidase. H2O2 was lysed rapidlyrelated genes were identified and their expression patterns werestudied. The 61 genes were divided into seven clusters based on the by the extracellular fluid even after catalase and myeloperoxidase

inhibition. Levels of H2O2 in wound fluid remained low and unchanged.similarities of their expression patterns. Forty-five genes showed anupregulation of expression at day 3. Included in this group were several

A major portion of supplemental oxygen that is delivered to woundscytokines (IL-1�, IL-6, IL-13), growth factors (KGF, HGF, TGF�-IIR),MMPs and TIMPs. Most of these genes return to their original level of is used to form intracellular oxidants. Enhanced bacterial killing is one

known consequence and wound angiogenesis is likely another. Multipleexpression at day 7. Twenty- two genes show a further increased levelof expression at day 7 (IL-15, HGF, KGF). Conclusions: The parallel extracellular antioxidants exert competent protection.expression patterns of the genes in each of the 8 clusters suggest thepossibility of those genes having common regulatory pathways. Several 86.cytokines, growth factors and protein turnover genes are expressed WOUND CARE QUALITY IMPROVEMENT TEAM: A VITALin concert during corneal wound healing. CR � none support by NIH RESOURCE FOR PRESSURE ULCER PREVENTION, COSTEY 05587. CONTAINMENT, AND QUALITY IMPROVEMENT IN ACUTE

HOSPITAL CARE.84.

Pozez,A.L.,Davis, K.,Blankenship, J.,Creehan, M.S., Hudson,K., Lucas,DIFFERENTIAL PROTEINASE EXPRESSION IN FETAL VERSUSV.S., and Keitz, J.ADULT MOUSE SKIN WOUNDS.The Wound Healing Center and MCV Hospitals, Division of Plastic andReconstructive Surgery, Medical College of Virginia/VirginiaE. Huang, E. Island*, H. Wu, D. Warburton, K. Anderson, and T-L. Tuan.

Department of Surgery, Children’s Hospital Los Angeles, Los Angeles, Commonwealth U, Richmond, VA.CA; Department of Surgery, University of California – Davis, CA.

The MCVH Wound Care Team, a collaborative resource to care forhospitalized patients with acute and chronic wounds, has focusedEarly fetal skin responds to injury via a process that results in scarless

repair of the damaged surface, a response different from the scar efforts towards reduction of hospital acquired pressure ulcers in ourinstitution. Initially to define the problem a chart review of 30,631formation seen in adult wounds. Proteolytic degradation of the

connective tissue extracellular matrix (ECM) is an essential feature of patient admissions from 1995-1996 was undertaken and 42 nosocomialulcers were identified. Data collection included diagnosis, age, LOS,wound repair and remodeling; however, this process has not been well

studied in fetal wounds. Comparing freshly isolated skin fibroblasts DRG, specialty bed use, and risk assessment, as well as evaluation oftotal charges, cost, reimbursement and lost revenue. Total charges forfrom fetal and adult mouse skins, we previously showed that there is

a difference in the expression and modulation of ECM proteases and that group, with LOS of 8-113 days were $5,664,666.72. Using Medicarereimbursement rates as the comparison for uniformity, we estimatedinhibitors during different developmental stages (Island et al., Wound

Rep Reg 7:467, 1999). An in vivo model in which cutaneous skin wounds a per patient loss of $21,356 over actual cost after average per patientpayment of $59,567.were induced to fetal mice in utero was used to study the process of

rapid fetal injury repair. These results were compared with similarOver the subsequent five years the Wound Care Team has focused itswounds induced in adult mice. The proteases involved in wound matrix

remodeling were studied using immunohistochemistry of injured mission, goals and objectives toward wound management,standardization of risk assessment, documentation, specialty bed usetissues. Results of in vivo wounding showed fetal skins of E15 mice

to re-epithelialize within 12 hours, faster than previously reported. and staff and resident education. The hospital has experienced a

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 161

The development of peri-implant capsules is associated with precedingreduction in overall noscomial pressure ulcer rate from 9.5% to 7.1%.The nosocomial rate was reduced from 25.7% to 21% in ICUs And from mast cell degranulation and subsequent persistent hyperplasia.

Therapeutic suppression of mast cell activity may help to prevent the6.2% to 4.8% in the adult medical-surgical areas.development and progression of peri-implant fibrosis. Further researchusing human tissue is indicated.We have repeated a chart review of 30,225 patient admissions from

1999-2000, identifying 66 patients with nosocomial pressure ulcers(*p < 0.025 compared with control).having LOS of 9-294 days. Total charges were $10,676,551.99. Estimated

per patient loss however reduced to $15,657 over actual costs withaverage per patient reimbursement rising to $68,865. We believe our 89.efforts have served to enhance quality of care delivered to patients DOES STEROID ADMINISTRATION INCREASE WOUND INFECTIONwith pressure ulceration and to focus our sustained goals to further AND/OR IMPAIR WOUND HEALING? EXPERIMENTAL STUDIES ONprevalence rate reduction while decreasing lost revenue. RAT.

H. Onodera, M. Onda, A. Tokunaga, T. Kiyama, T. Yosiyuki, and G.87.

Masuda.A MATHEMATICAL MODEL OF THE EFFECTS OF TRANSFORMINGDepartment of Surgery, Nippon Medical School, Tokyo, Japan.GROWTH FACTOR BETA ON EXTRACELLULAR MATRIX

ALIGNMENT IN DERMAL WOUND REPAIR.The detrimental effects of steroids on wound infection and woundhealing are well recognized. However, the mechanisum is poorlyJ.C. Dallon. Department of Mathematics, Brigham Young University,

Provo, UT, J.A. Sherratt, Department of Mathematics, Heriot-Watt understood. A rat model for long-term preoperative and perioperativecorticosteroids was used in the study. The mortality rate of peritonitisUniversity, Edinburgh U.K., P.K. Maini, Centre for Mathematical Biology

Mathematical Institute, University of Oxford, Oxford U.K. induced by CLP(Cecum Ligation Puncture) and bursting pressure ofthe colonic anastomosis were investigated in cortison treated rats.

In order to understand the anti-scarring properties of TGF-beta, we Materials and Methods. Steroid administration and peritonitis; forty-three male Sprague-Dawley rats, weighting 180 to 220, were randomizedmust understand each of its individual effects on the wound process,

how they interact and eventually result in less scarring. To this end, into two groups. The steroid group received a time-release drug pallet(28mg/kg cortisone in 14-day release form) placed in the subcutaneouswe have developed a mathematical model of alignment in wound

healing which incorporates some of the effects of TGF-beta. In this tissue of the posterior. The control group received placebo. At 14-dayafter surgery, body weight, urine volume, 17-KS in urine, water andmodel we focus on the interactions between fibroblasts and the

extracellular matrix. We allow for time varying concentration of TGF- the volume of meal were measured. CLP was performed (23G, 18G?1,18G?2, 16G?2) at the 14 days after surgery, and the mortality rate wasbeta which can alter the motility, proliferation and collagen production

of the fibroblasts. We find that although these properties of the determined at the 96-hours. Wound healing study; twenty-eight maleSD rats randomized into steroid group and control group. Burstingfibroblasts can affect matrix alignment they seem to be of minor

importance and cannot explain the anti-scarring properties of TGF- pressure was measured at 5 and 7 days after colonicanastomosis.Results. Steroid administration and peritonitis; Bodybeta. However, we find that by changing fibroblast reorientation rates,

consistent with experimental evidence, the alignment of the weight and urine volume were significantly higher in the steroid groupthan in the control group. Blood cortisol and ACTH did not differregenerated tissue can be significantly altered and thus explain the

influence of TGF-beta on scarring. among two groups. The mortality rate was significantly higher in thesteroid group than in the control group increase of 16G?2 needlepuncture. Wound healing study; The bursting pressure of colonic88.anastomosis was not different in two groups at 5 and 7 days afterTHE KINETICS OF MAST CELLS IN THE DEVELOPMENT OF PERI-surgery. Conclusion. This study is thought to be high dose of steroidsIMPLANT CAPSULES.administration model. Increased rate of death induced by peritonitis

Ben Chew, Stuart McKirdy, Ian Naylor, David Sharpe. and no impairment of the healing of colonic anastomosis were observedPlastic Surgery and Burns Research Unit, University of Bradford, United in steroid treated animals.Kingdom.

90.Capsular contracture is the most common complication following ANALYSIS OF THE ROLE OF BASIC FIBROBLAST GROWTH DURINGsilicone prosthesis implantation, yet its aetiology remains unresolved. DUODENAL ULCER HEALING.Fibroproliferation in hypertrophic scars, lung, liver and sclerodermais associated with increased mast cell activity. The kinetics of mast Shinya Iida1), Masahiko Onda2), Akira Tokunaga2), Teruo Kiyama2),

Kaku Egami1), Shotaro Maeda3), 1)Department of Surgery, Tama-cells in peri-implant fibrosis was investigated using a rodent model.Nagayama Hospital, Nippon Medical School, 2)Department of Surgery,Nippon Medical School, 3)Department of Pathology, Tama-NagayamaOne silicone implant was sited in each of 28 Hooded-Lister rats within

the sub-dermal fascia of the left dorsal thoracic area. Animals were Hospital, Nippon Medical School, Tokyo, Japan.randomly sacrificed in groups of four on days 1, 3, 5, 7, 14, 30 and 60.Implants and surrounding tissues were excised en bloc to maximally There are many reports about the healing mechanism of peptic ulcer,

but it has not been clarified in details. We have paid attention to growthpreserve the in situ architecture of capsular and pericapsular tissue.Control tissue was obtained from the unwounded contralateral side. factors in the process of wound healing in gastrointestinal tract. It was

previously reported that basic fibroblast growth factor (bFGF) was anSpecimens were processed and analysed by conventional histology forcollagen (Herovici trichrome) and mast cells (toluidine blue). activator of cell proliferation, angiogenesis and formation of

granulation by stimulating fibroblasts. We now analyze the localizationImmunohistochemistry for smooth muscle alpha-actin was used todemonstrate myofibroblasts. Mean mast cell counts (number/mm2) of basic fibroblast growth factor (bFGF) and K-SAM (FGFR2), one of

the receptor of bFGF, and gene expression of bFGF in the duodenalwere determined using a standard method. Statistical analysis wasperformed using the Mann-Whitney U test. ulcer in order to elucidate the role of bFGF during duodenal ulcer

healing.A marked decline in mast cell numbers from control (89/mm2) was

We examined the localization and gene expression on the surgicalobserved on days 1 (36/mm2)* and 3 (41/mm2)*. Normal mast cellnumbers were restored by day 7 (88/mm2), coinciding with the first specimens from patients with duodenal ulcer who underwent distal

gastrectomy for perforation, uncontrollable bleeding, or stenosis ofappearance of capsular organization and myofibroblast expression.Capsular development, myofibroblast expression and mast cell the duodenum by use of immunohistochemistry and in situ

hybridization. Immunostaining was carried out on dewaxed paraffin-numbers increased progressively until day 30 (186/mm2)*. Capsulararchitecture, myofibroblast expression and mast cell counts remained embedded sections, and the sections were reacted with antibodies

raised against human bFGF (Oncogene Research Products, Cambridge,unchanged between days 30 and 60.

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001162 Meeting Schedule and Abstracts

MA), and K-SAM (Genetics Division, National Cancer Center, Japan). glycosaminoglycan (GAG) copolymer with controlled porosity anddegradation rate that promotes tissue ingrowth without causing anWe performed in situ hybridization on the same sections with human

bFGF oligonucleotide probe. inflammatory response. The temporary epidermal substitute layer ismade of silicone and functions to immediately close and prevent

K-SAM (FGFR2) protein was localized widely in the duodenal sections, moisture loss from the wound. After neodermal formation (in 14 to 21days), the silicone layer is removed and a very thin meshed epidermalwhile protein localization and gene expression of bFGF was observed

mainly at the duodenal epitheliums and Lieberkuhn’s glands and autograft is applied. A next generation technology, under development,is based on providing specific cellular adhesion and migration signalsfibroblasts nearby active inflammatory regions in the sections.by covalently binding specific peptide analogues of the cell adhesionsequence Arg-Gly-Asp (RGD) to the collagen-GAG copolymer. Peptide-Based on these results, we conclude that bFGF plays roles on the

healing process of duodenal ulcer at the early phase. enhanced TRTs demonstrated a faster neodermal formation than thecurrent templates in a full thickness excisional wound healing modelin guinea pigs. Analysis of histological sections immuno-stained for91.

alpha smooth muscle actin of the neodermis formed in these peptide-A COMPARISON OF BECAPLERMIN GEL AND MYOCUTANEOUSenhanced templates indicates a lack of myofibroblast formation, whichFLAP CLOSURE IN THE TREATMENT OF TWO SEPARATE ISCHIALitself is an indicator of potential future wound contraction and scarring.PRESSURE ULCERS IN THE SAME PATIENT.These results suggest that improved dermal regeneration via RGD

Thomas Serena and Carrie Corbran. Penn North Center for Advanced signaling may provide a significant clinical advantage.Wound Care, Warren, PA. and Gannon University, Erie, PA.

93.The development of pressure ulcers in spinal cord injured patients can REDUCED FOREIGN BODY RESPONSE IN SPARC-NULL MICE.lead to significant morbidity and mortality. Traditionally, these ulcershave been treated with myocutaneous flap surgery. This is an expensive P.Puolakkainen, A.Bradshaw, T.R.Kyriakides, A.Lehman, R.Brekken,

P.Bornstein, E.H.Sage. The Hope Heart Institute and University ofprocedure with its own associated morbidity. Recently, human plateletderived growth factor &#8211; BB becaplermin (Regranex) has been Washington, Seattle, WA.demonstrated to be efficacious in the treatment of chronic pressureulcers. SPARC (secreted protein acidic and rich in cysteine), a matricellular

glycoprotein modulates the interaction of cells with the extracellularWe present the case of a twenty-five year old C-5 quadriplegic who matrix. SPARC is expressed in cutaneous wounds, in healing bowel

anastomoses, and in areas of rapid cell proliferation. Recently,developed a Grade 4 right ischial decubitus ulcer. At that time he hada 9 � 6 � 4 cm. Grade 4 right ischial ulceration. He was treated with accelerated cutaneous wound healing was reported in SPARC-null

mice. The purpose of the present study was to evaluate whether SPARCsaline dressings and nutritional supplementation preoperatively. Heunderwent an uncomplicated myocutaneous rotational flap without influences the foreign body response to implanted biomaterials.

Silicone discs and millipore filters (type HA, mixed cellulose ester,complication. He spent a total of four weeks in the hospital for off-loading of the surgical site (three weeks were spent in an air bed). He pore size 0.45um) were implanted subcutaneously into wild-type and

SPARC-null mice and were sampled after 4 weeks. Encapsulated tissueshas remained healed. Three years later the patient returned with a newischial decubitus ulcer, only this time it was on the opposite side. were prepared for histological analysis. Capsule thickness, vascularity,

inflammatory reaction, and the number of giant cells were assessedBecause of the lengthy hospitalization associated with his previousflap closure, the patient did not wish to undergo flap closure for this for all implants. The mean relative( � SD) capsule thickness in wild-

type mice was 3.6 � 0.8 for silicone implants and 3.0 � 1.8 for milliporeulcer (5 � 4 � 4 cm.). For this reason the ulcer was treated withtwice daily saline dressings and daily application of becaplermin filters. In SPARC-null mice the values were 1.8 � 0.3 and 1.3 � 0.4,

respectively. The index of vascularity in wild-type mice was 3.2 � 0.6(Regranex). The ulcer was 90% healed in eleven months and reachedcomplete healing at eighteen months. At one year follow up he remains for silicone implants and 2.2 � 1.2 for millipore filters. Corresponding

values in SPARC-null mice were 1.1 � 0.4 and 0.7 � 0.3, respectively.completely healed.Foreign body giant cells were more abundant in wild-type mice in bothimplant groups. The collagenous capsule was analyzed by PicrosiriusDespite the fact the becaplermin treated ulcer required a considerably

longer period of time to heal, the patient was far more satisfied with red staining and electron microscopy. Collagen fibers were found tobe smaller and more disorganized in SPARC-null mice. Significantthis treatment regimen. This was primarily because he avoided the

lengthy hospital stay. In addition, in comparing the cost between the immunoreactivity for SPARC was observed in the cells and matrix thatcomprise the capsules surrounding the implants. The reduced capsuletwo treatment regimens, the total cost for the flap surgery was

$24,053.00; whereas, the total cost for the becaplermin treatment was thickness and reduced vascularity indicative of the foreign bodyresponse in SPARC-null mice implicate SPARC as an important$2405.00 which includes three prescriptions of becaplermin.mediator in the encapsulation of implanted biomaterials.

The use of becaplermin gel in the treatment of pressure ulcers in spinalcord injured patients may lead to greater patient satisfaction in selected 94.

cases. It also has the advantage of being far less costly when compared AN UNUSUAL PRESENTATION OF ADENOCARCINOMA IN A POST-to traditional myocutaneous flap closure. TRAUMATIC MALE BREAST WOUND.

Robin R Hamlin, Scott D Green, Anil Punjabi, and Subhas C Gupta.92.

Division of Plastic Surgery, Loma Linda University, Loma Linda,DEVELOPMENT OF AN IMPROVED TISSUE REGENERATIONCalifornia.TEMPLATE FOR DERMAL WOUND REPAIR.

Adenocarcinoma of the male breast is uncommon and typically presentsJames O. Tolley, Timothy I. Malaney, Rose Bellantoni, Juerg F. Tschopp,Frederick Cahn, and Michael D. Pierschbacher. Integra LifeSciences as a painless mass or nipple discharge. In the US there are only

approximately 1,000 cases a year, and treatment is usually extrapolatedCorporate Research Center, San Diego, CA.from breast cancer treatment in women. The poor prognosis anddecreased survival rates in men compared to women is likely due toWe have used a guinea pig dermal wound model to predict the clinical

performance of a modified form of INTEGRA�8482; Artificial Skin. delays in diagnosis and treatment. This paper describes an unusualpresentation of adenocarcinoma in a male breast wound and a meta-The clinical objective is a skin replacement procedure with more rapid

dermal regeneration, reduced scarring, and a reduced need for analysis of previously reported post-traumatic wound male breastadenocarcinoma. Our discussion includes a summary of the diagnostic,autologous donor tissue. INTEGRA�8482; Tissue Regeneration

Template (TRT) is an FDA-approved bilayer membrane skin therapeutic, and reconstructive options for treating patients with thesewounds.replacement system that permanently replaces injured skin with

functional autologous tissue. The product is currently used as aA 33 year old male initially presented 5 years after sustaining a gunshottreatment for full thickness or deep partial thickness burns. The dermal

regeneration layer is composed of cross-linked collagen- wound to the left chest. He developed ulceration and progressive

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 163

Much interest has recently been shown in elucidating the signals whichbreakdown of his scar with wound enlargement. Initial treatmentinvolved split thickness skin grafting on two separate occasions that regulate both skin development and wound repair. In contrast to prior

reports using less sensitive degenerate primers, we have utilized gene-resulted in complete graft loss. The wound continued to enlarge anda biopsy was performed to make the diagnosis of breast specific primers and demonstrated by PCR an unexpectedly widespread

expression pattern of most of the wnt family members in postnataladenocarcinoma. The wound had progressed to 60 by 68 centimetersand encompassed half of the patient’s torso at that time. skin and wounds. As wnt morphogens are powerful inducers of

architecture and form in the developing embryo, we believe they willEarly recognition and treatment of male breast cancer is essential and be important in restoring dermal architecture and inducing regenerative

processes during normal wound healing.can significantly decrease the morbidity and mortality associated withthis disease. Education and increased awareness are necessary toimprove both prognosis and survival rates. 97.

COMBINATION GENE THERAPY WITH BASIC FIBROBLASTGROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH95.FACTOR SYNERGISTICALLY AUGMENTS FLAP SURVIVAL.ENDOTHELIAL CELL RESPONSE TO ISCHEMIA.PY Liu, K Liu, E Badiavas, IG Summerhayes. Department of PlasticN. Puzziferri, L. Humphrey, T. K. Hunt, and N. Boudreau. University of Surgery, Lahey Clinic, Burlington, MA.California, San Francisco, CA.

Wound healing may be an important application for gene therapy, andIschemic tissues and wounds are characterized by hypoxia, increased we have previously demonstrated prefabrication of reconstructive flapslactate, and oxidants. Hypoxia and oxidants elicit a family of heat with lipofection-mediated gene transfer of VEGF. We now haveshock proteins (HSPs). The effect of lactate is unknown. We postulate evidence that combination gene therapy, using two of the earliestthat these conditions elicit a coordinated angiogenic response from growth factors elaborated by a healing wound, is also efficacious.endothelial cells that includes hsp47, the collagen chaparone, withvascular endothelial growth factor (VEGF) and collagen. One week prior to raising 3 � 10 cm dorsal random pattern skin flaps

in female Sprague-Dawley rats, cDNA encoding b-FGF was combinedPrimary cultures of human microvascular endothelial cells (HMVEC) with cDNA encoding VEGF-165, complexed to Lipofectin, and injectedwere exposed to heat shock (43*C), hypoxia (15mmHg O2), lactate into the area. Following flap elevation, animals were observed for one(15mM), and hydrogen peroxide (H2O2, 2mM). After 8-16 hours week, and flap survival was ascertained via digitized surface planimetry.recovery Northern and Western blotting were performed. Histological and molecular analysis was then carried out.

Collagen and VEGF messenger RNAs were increased by heat shock Our results document enhanced survival of flaps transduced with bothas well as high lactate. Hsp47 was increased by lactate, and decreased bFGF and VEGF relative to plasmid controls (64.24% vs. 50.94%, n �by hypoxia and exposure to H2O2. 5, p < 0.009), and also relative to either bFGF alone (52.14%, n � 10)or VEGF alone (57.70%, n � 10). PCR amplification of flap tissueHMVEC respond to the environment of injury with a coordinated confirmed expression of both VEGF and FGF cDNA.response that involves hsps, collagen and VEGF. Lactate may be a

better stimulus for angiogenesis than hypoxia. This synergism leads us to conclude that combination gene therapy isfeasible and may represent a logical extension of the work being done

96. in single gene systems.MORPHOGENS AND THEIR RECEPTORS ARE WIDELY EXPRESSEDIN POSTNATAL SKIN AND WOUNDS. 98.

TOPICAL PLATLET DERIVED GROWTH FACTOR ENHANCESTamara N. Elias, Robert D. Galiano, Mary Armour and Geoffrey C. WOUND CLOSURE IN THE ABSENCE OF WOUND CONTRACTIONGurtner. IN PATIENTS.Institute of Reconstructive Plastic Surgery, NYU Medical Center, NewYork, NY. Bruce M. Freedman, H. Paul Ehrlich. Plastic Surgery Associates of

Northern Virginia; McLean, VA and Div. of Plastic and ReconstructiveThe wnt family of morphogens, consisting of at least 16 different Surgery; Hershey Med. Center, Hershey, PA.members and 10 receptors, is important in a wide range ofdevelopmental events. In order to rigorously define the role of this Recombinant human Platelet Derived Growth Factor (rhPDGF-BB;

Regranex) has been shown to promote wound healing in animals andfamily of morphogens during both adult and fetal skin wound healing,we first set out to definitively determine their expression pattern in patients. However, in the in vitro FPCL contraction model, PDGF failed

to enhance FPCL contraction through a mechanism involving theadult skin and wounds.reorganization of collagen fibrils (J Cell Physiol 185:432-439). A reviewof the literature showed conflicting reports about PDGF enhancingThe rat was chosen as an animal model because of its potential for

performing fetal wound healing experiments in future experiments. wound contraction in in vivo models. Here we investigated the effectof topically applied PDGF in a human model of wound contraction.Using sequence-specific primers when available, and primers designed

against mouse and human wnt mRNA sequences at regions of perfect Compared to loose skin lab animals, the importance of woundcontraction in the closure of human excisional wounds is restrictedhomology, most of the rat wnt genes were cloned. Four of the wnt

receptors (frizzled1-4) were also cloned. The identity of each cDNA to a few anatomical locations. One such location is the skin behindthe ears, where a full thickness wound will close by wound contractionproduct was verified by DNA sequencing. For the expression analyses,

RNA was harvested from unwounded rat skin as well as from 6-mm in about 3 weeks. In 2 patients undergoing elective surgery, a 0.5 cm2full thickness punch wound was made behind each ear. Each left post-dermal punch wounds at days 3, 7 and 14 days postwounding. The

RNA was treated with DNAse to remove contaminating genomic DNA articular wound was treated daily with an application of rhPDGF gel.Each right post-articular wound was treated daily with petroleum basedand cDNA was then synthesized for use in RT-PCR reactions.gel. All wounds were dressed with saline moistened gauze. The left-side-treated wounds were completely closed by day 15. The right-side-Most of the wnt genes were expressed in wounds and skin. Specifically,

wnt-2a, wnt-2b, wnt-3, wnt-4, wnt-5a, wnt-6, wnt-10a, wnt-10b, wnt-11 control wounds required 19 days for complete closure. All healedwounds were harvested at day 20 and the tissues processed for lightand wnt-16 were expressed in unwounded skin and wounds. The wnt

genes that were not expressed were wnt-1, wnt-5b, wnt-7a, wnt-7b, microscopy. By H & E staining the PDGF treated wounds showed agreater cell density beneath the intact re-epithelialized surface. Mostwnt-8b and wnt-15. All 4 frizzled receptors studied were detected in

both skin and wound samples. As this experiment set out to solely of these cells appeared to be fibroblasts. Polarized light optic viewingof Sirius red stained treated specimens showed a pattern of finedetect the presence or absence of these genes, no attempt was made

to quantify the absolute or relative amounts of different wnt members collagen fibers birefringence, which is characteristic of maturinggranulation tissue, deposited throughout the healed wound site. Thein skin or wounds.

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001164 Meeting Schedule and Abstracts

control-healing site showed a low density of cells. The birefringence euthanized since seizures lasted for more than 6 hours. In all survivingrats, locomotion was normal 24 hours after FS application.pattern of more than 90% of the collagen fibers within the biopsies

was that of intact dermal collagen fibers, the hallmark of wound closureIn conclusion, FS containing t-AMCA may cause seizures if appliedby the process of wound contraction. In contrast, the PDGF treated

wounds that closed faster than controls, healed by re-epithelialization topically to the cortex whereas FS containing aprotinin do not causeany acute convulsions. Tranexamic acid most likely is the activeand filling-in with scar. It is proposed that the enhancement of wound

closure by re-epithelialization and limiting wound contraction are substance responsible for the epileptic action of FS containing t-AMCAsince the extent of convulsive behaviors increased with increasingcharacteristics of the locally applied PDGF.dosage of t-AMCA. Thus, FS containing t-AMCA should not be appliedto the cortex.

99.

101.

HIGH MORBIDITY AND HOSPITAL COSTS OF STAGE IV PRESSUREULCERS.

Brem H, Vladeck B, Nierman D, Bell D, Kapil-Pair N, Kaplan D, andHollier L. Department of Surgery, Mount Sinai Medical Center, NewYork, NY.

The purpose of this research is to establish a method of total costanalysis for patients with stage IV pressure ulcers in a hospital setting.By following total costs/charges directly related to the patients, thecost of nonhealing, morbidity and mortality are significantly higherthan previously reported. We retrospectively analyzed the charges forpatients who developed pressure ulcers in the hospital (n � 11), butpresented with an unrelated clinical problem. In comparison, wefollowed patients who had recurrent admissions for communityacquired pressure ulcers (n � 8). Only charges directly related to theconsequences and complications of a non-healing wound werecalculated. Hospital acquired pressure ulcers cost $225,615 per patient(with one admission per patient) and community acquired pressureulcers cost $180,693 per patient (with four admissions per patient). Incontrast to hospital acquired pressure ulcers (which were by definitiononly one admission per patient), the community acquired pressureulcers averaged four admissions per patient. The primary cost ofhospital acquired pressure ulcers was renal dialysis, secondary to sepsisfrom the wound. No patients were in renal failure or required dialysisprior to hospitalization. The incidence of the morbidity complicationswas not associated with the patients’ initial diagnosis (e.g. pneumonia).Although the morbidity and overall costs per admission for communityacquired pressure ulcers are significantly less, the overall cost to societyremain exceptionally high. The direct hospital costs of pressure ulcers

100. often exceed hundreds of thousands of dollars per patients. In theEPILEPTIC SEIZURES INDUCED BY FIBRIN SEALANTS future, early recognition and treatment may prevent these costs. WeCONTAINING TRANEXAMIC ACID. hypothesize that by evaluating the total cost of treatment of a patient’s

wound; millions of dollars and significant patient morbidity in anyMichael Schlag, Rudolf Hopf, Heinz Redl. Baxter AG, Vienna, Austria; tertiary care medical center will be saved per year.Boltzmann Institute for Traumatology, Vienna, Austria.

102.The aim of this study was to elucidate whether fibrin sealants (FS), RAPID HEALING RATES OF ELDERLY PATIENTS WITH DIABETICwhich are frequently used to support wound healing, evoke seizures FOOT ULCERS, VENOUS STASIS ULCERS AND PRESSURE ULCERS,in a rat model. Male rats (350-400 g) were randomly assigned to one AFTER APPLICATION OF HUMAN SKIN EQUIVALENT.of 5 groups. In groups 1-4 (n � 24), a right craniotomy was performedand the dura was resected, while no craniotomy was carried out in Brem H, Baskin-Bey E, Kapil-Pair N, Kaplan D, Balledux J, Hollier L.

Mount Sinai Medical Center, New York, NY.group 5 (n � 4). FS containing either aprotinin (group 1), 0.5 mg/mltranexamic acid (t-AMCA; group 2), 5 mg/ml t-AMCA (group 3), or 45

The use of established surgical and wound-healing techniques inmg/ml t-AMCA (group 4) were applied to the cortex. Followingapplication, EEGs and general behavior were continuously recorded conjunction with Human Skin Equivalent (HSE - consisting of cultured

keratinocytes and fibroblasts on a type I collagen) will result in rapidduring the first 60 min and 120 min, respectively. Rats were allowedto recover and locomotion was assessed 24 hours after FS application. healing of chronic wounds in the elderly. Forty consecutive patients

between 65 and 102 years who had nonhealing venous stasis ulcersRats were euthanized thereafter.(Figure A), diabetic foot wounds, sacral, trochanteric or ischialpressure ulcers were treated. Each wound was between 1 and 15 cmAt the time point of application, all rats were in deep anesthesia. In

group 5, the EEG recordings during the first 60 min post application in length. After surgical debridement, all patients received a singleapplication of human skin equivalent. The skin graft was applied usingshowed a gradual transition from slow regular waves (1-1.5 Hz) to fast

irregular waves ( > 8 Hz) indicating recovery from anesthesia. Rats in interrupted 5-0 absorbable sutures with a 1-millimeter distance betweenthe applied new skin and the excised ulcer edge. Overall, 29 out of 40group 1 showed very similar EEG recordings and did not express any

convulsive behavior during the 2 hr observation period. Application of elderly patients went on to 100% healing (figure B). Of the elevenpatients whose wounds did not heal, each had a wound that hadFS containing different concentrations of t-AMCA resulted in different

grades of convulsive behavior. In group 2 (n � 6), one rat showed 3 extended to bone, or Methicillin Resistant Staphylococcus was alreadypresent at the time of HSE application. Elderly patients do not havebrief episodes of jerk-correlated convulsive potentials whereas the

other 5 rats showed no abnormal EEG recordings. In group 3 (n � significant clinical impairments to wound healing, but ratherexperience a higher incidence of the underlying pathogenesis of chronic6), 5 rats showed jerk-correlated convulsive potentials and 1 rat even

developed generalized seizures. However, convulsive behavior ceased ulcers, e.g. venous reflux, diabetes and being bed bound Seventy-twopercent of 40 consecutive elderly patients with chronic nonhealingafter app. 100 min and all animals survived. In group 4 (n � 6), all

rats developed jerks and generalized seizures. Four rats had to be wounds (including venous stasis ulcers, diabetic foot ulcers, and

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 165pressure ulcers) that were treated with human skin equivalent resulted objective was to confirm efficacy, safety and application guidelines of

Graftskin, in patients with diabetic neuropathic foot ulcers.in 100% healing of their wounds. Human Skin Equivalent was successfulby accelerating wound healing and releasing multiple growth factors

This was and open-label, multi-center study of 13 adult patients withinto the wound, rather than acting as a traditional skin graft. Wespeculate that elderly patients’ wounds will heal if treated with diabetes with plantar neuropathic ulcers. The ulcers had to be present

for at least 4 weeks prior to study participation, thus demonstratingaggressive debridement and HSE before morbidity and mortality thatmay be related to the wound develop. unresponsiveness to conventional therapy. The primary efficacy

variable was time to 100% wound healing. Healing was defined as fullepithelialization of the wound with the absence of drainage.103.

THE EFFECT OF TRANEXAMIC ACID MIXED IN FIBRIN SEALANTDuring the 12-week treatment period up to 3 applications of GrafskinCLOTS AND SOLUTION ON ADHESION, PROLIFERATION AND CELLcould be applied to the target ulcer with a minimum of 2 weeks betweenVIABILITY OF NEURONAL AND NON-NEURONAL CELLS.applications. All patients received good wound care including surgical

Steve Cox, Marietta Cole, Samia Mankarious and Nabil Tawil, Wound debridement, off-loading of the ulcer, and infection evaluation. AllMgt. Group. Baxter Healthcare Corp, Duarte, CA. patients had to continue with acceptable off-loading and standard

wound care procedures until the follow-up safety visit.Fibrin sealant products, commonly used for hemostasis in various typesof tissue, normally contain a fibrinolysis inhibitor. Tranexamic acid Graftskin achieved complete healing in 9 of 13 (69%) within the 12(tAMCHA) is a common and effective fibrinolysis inhibitor and is used weeks of the study. Seven of these patients (54%) achieved completein some currently available fibrin sealant products. Recent studies of healing after 7 weeks. An average of 1.5 applications were required tofibrin sealant products containing t-AMCHA indicate that it may be achieve complete healing.responsible for various adverse reactions when used in neurosurgery.To determine a possible mechanism, we examined the effect of tAMCHA There were no adverse events at the treated ulcer sites and none ofon the behavior of proliferative cells (glial and fibroblasts) and non- the reported adverse events were suspected to be related to Graftskin.proliferative cells (neuronal). We found that tAMCHA, incorporatedinto fibrin clots, did not effect the initial cell adhesion of either This open study supports the pivotal data approved by the FDA forproliferative or non-proliferative cells. In contrast, tAMCHA in solution the treatment of diabetic neuropathic foot ulcers. Graftskin is safe andreduced the initial cell adhesion of all cell types to coated culture effective in the treatment of diabetic neuropathic foot ulcers. Additionaldishes. We also found that a higher concentration (300-450mM) of studies need to be performed to substantiate application guidelines.tAMCHA reduced cell proliferation of proliferative cells on coateddished, but seemed to enhance their proliferation on fibrin clots. In

106.addition, a high concentration of tAMCHA led to neuronal death andUSE OF A SELF-ASSEMBLED RECONSTRUCTED SKIN FOR THEreduced the number of non-proliferative neuronal cells on both clotsHEALING OF A VENOUS LEG ULCER.and coated dishes. Our model suggests that tAMCHA modulates cell

adhesion and possibly affects cell signaling, which ultimately influences Marie-Helene Rochon, Nathalie Dube, Herve Genest, Francois A. Augercell proliferation and cell death. Thus, a high concentration of tAMCHA and Lucie Germain. Laboratoire d’Organogenese Experimentale,should not be used as a fibrinolysis inhibitor in fibrin sealant products CHAUQ Hopital Saint-Sacrement, Departement de chirurgie, Universiteused in surgery applications. Laval, Quebec, Canada.

104. The main goal of this study was to use an autologous self-assembledTHE USE OF SANTYL COLLAGENASE IN THE TREATMENT OF reconstructed skin to accelerate the wound closure of a difficult toDIABETIC FOOT ULCERS: A DOUBLE BLIND PROSPECTIVE heal venous leg ulcer.RANDOMISED STUDY.

The autologous cells used to produced this reconstructed skin wereKenneth N. Dolynchuk, and Merv Low. Department of Surgeryisolated from a small (1 cm2) cutaneous biopsy. The fibroblasts were,University of Manitoba, Winnipeg, Manitoba.cultured in the presence of ascorbic acid to form sheets. Threefibroblasts’ sheets were superposed to form a reconstructed dermis.The use of Santyl collagenese has been reported to be of benefit inKeratinocytes were seeded on top of the reconstructed dermis andlower extremeity ulceers. The purpose of this study was to determineallowed to mature at the air-liquid interface. The histological andif a mixture of Santyl and bacitracin alone, as measured by the rateultrastructural features of this self-assembled skin are similar to thoseof wound healing. The protocol included diabetics with foot ulcersof a normal human skin. The self-assembled reconstructed skinwhich were not septic and followed best practices overall for wounddevelopped in our laboratory does not contain any exogenous orcare, including offloading. The data were measured and recorded usingsynthetic material.digital images and the VeMD Measurement program to calculate the

Margolis equation. Values were taken ar 1, 3 and 6 weeks and theThe reconstructed skin was placed on the debrided venous leg ulcerspatients’wound examined. The results in ten patients to date showedand was maintained in place with a compressive dressing. Until woundthat the patients were not adversely affected by the use of Santyl andclosure, a new self-assembled skin was placed on the ulcer each week.bacitracin mixed 50:50 when compared to bacitracin alone. In fact theWeekly evaluation of the wound were performed by the physician andoverall rate of woundhealing was approximately 0.05 cm/week fasterthe nurse.as measured. We conclude that the use of Santyl with bacitracin is

saffe and effective as a means of preparing the wound bed in diabeticThe self-assembled reconstructed skin was easy to manipulate withfoot ulcers as compared to the use bacitracin.forceps and scissors as assessed by the physician. The treated patienthad two venous leg ulcers underneath his left intern maleola. A total

105.of three applications were necessary to heal both ulcers. Wound healingEFFICACY AND APPLICATION GUIDELINES OF GRAFTSKIN FORwas observed after the first week of treatment. The application of theDIABETIC NEUROPATHIC PLANTAR FOOT ULCERS.self-assembled reconstructed skin lessened the pain caused by theulcers. Until now, three months after the end of his treatment, noL. Lindlsey, E. Hornby, K. Marshall. Novartis Pharmaceuticals

Corporation, East Hanover, NJ. reccurrence of the ulcers was observed.

Approximately 16 million individuals in the United States have diabetes. The self-assembled reconstructed skin is composed of healthy cellsembedded in a complex extracellular matrix. The biological signalsDiabetic foot ulcers affect 15% to 20% of this population and account

for 86,000 amputations per year. The cost associated with this condition from the reconstructed skin favored the closure of a difficult to healwound in a short period of time. In this pilot study, five other patientsexceeds $ 1 billion per year. Newer treatment modalities including

growth factors and tissue engineered skin, when combined with proper will be treated with this novel living skin substitute produced by theself-assembly approach.wound care, have shown improvements in outcomes. The primary

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001166 Meeting Schedule and Abstracts

107. 109.

COLLAGEN-POLYVINYLPYRROLIDONE PROMOTES HUMANWOUND HEALING THROUGH CYTOKINE DOWNMODULATION.

Ricardo Rodrıguez-Calderon, Janette Furuzawa-Carballeda, AlbertoCorchado and Edgar Krotzsch*. Department of Cell Biology. Institutode Investigaciones Biomedicas, UNAM; *Biomedical Research Division,CMN; 20 de Noviembre, ISSSTE, Mexico City, Mexico.

Nowadays several cytokines and extracellular matrix (ECM)components are used to promote wound healing. Our group has beenworking in the searching of collagen-polyvinylpyrrolidone (collagen-pvp) mechanism of action, since it has demonstrated to improve woundand fracture healing. Two centimeter incissional wound was made onthe interior arm of eleven volunteers and a thin piece of the skin wasobtained as a control. Patients were divided in two groups: a placebogroup who received citrate solution in the wound and the experimentalgroup treated with collagen-pvp in citrate solution. Both groups weresutured with nylon. Biopsies were taken at 7 and 28 days post-surgeryfrom different individuals. Wound and skin fragments were processedby histological and immunohistochemical methods. Clinically nosignificant differences were detected between groups. But structurally,collagen and elastin fibers showed a better pattern array in the collagen-pvp treated group although MMP-1 and TIMP-1 levels did not changed.A significant diminution of PDGF-AB, TGF-�1 and TNF-a expressionby spread and blood vessels cells, at 7 and 28 days after surgery wasobserved. On the other hand, VCAM-1 diminished meanwhile VLA-4was increased at day 7, which indicates a possible roll of cytokines inthe control of diapedesis although leukocytes keep the activated state.In conclusion, collagen-pvp modulates inflammation in reparative 110.processes and it is supported by previous data where proinflammatory/fibrogenic cytokines were downmodulated in dermal fibrosis andinflammed synovial tissue by the treatment with the biodrug.

This study was partially supported by Aspid, S.A de C.V.

108.

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 167

We suspect that 2.5SNGF, a biologically active subunit of NGF improves111.

wound closure kinetics by promoting epithelialization. Further studiesmust determine whether inflammation or angiogenesis are acceleratedin early wounds.

113.

IMMEDIATE-EARLY INDUCTION OF c-JUN IN THE PANCREAS OFMICE AFTER BURN INJURY.

Kiho Cho, Lee K. Adamson, and David G. Greenhalgh.Shriners Hospitals for Children Northern California and Departmentof Surgery, University of California at Davis, Sacramento, CA.

A large burn often causes distant organ failure, such as acute respiratorydistress syndrome, in addition to skin damage. The cellular andmolecular signaling events leading to multiple organ failure after burninjury has not been well characterized yet. We hypothesize that burninjury induces pathophysiologic changes in the pancreas and some ofthese changes are initiated by the orchestrated regulation of immediate-early response genes (e.g., transcription factors). In this study, weinvestigated the effect of burn injury on the expression of c-Jun andc-Fos in the pancreas.

C57BLKS/J female mice were subjected to 18%TBSA full-thickness burnand tissues were harvested at different time points (no burn controls,3 hours to 21 days). Immunohistochemistry and Western blot usingrabbit polyclonal antibody were performed to determine the regulationof c-Jun and c-Fos expression in the pancreas in response to burninjury.

Immunohistochemical analysis of c-Jun and c-Fos regulation revealedthat the nuclear staining of c-Jun in the pancreas, but not c-Fos, wasrapidly and transiently up-regulated 3 hours after burn injury andreturned to the basal level thereafter. This immediate-early up-regulation of c-Jun in the pancreas after burn injury was confirmed by

112. Western blot analysis.NERVE GROWTH FACTOR ACCELERATES WOUND HEALING INGENETICALLY DIABETIC MICE. These findings and our previous study of the differential regulation of

c-Jun in the liver and lung after burn injury suggested that c-JunPM Muangman, ML Spenny, J Anthony, JE Olerud*, NS Gibran. participates in the immediate early cellular signaling events leading toDepartments of Surgery and *Dermatology, University of Washington, the functional changes in distant organs (e.g., pancreas, liver) of miceSeattle, WA. after burn injury. Further characterization of the downstream signaling

events, as well as the exocrine and endocrine functions of pancreas,Diabetic patients have reduced numbers of cutaneous nerves, which after transient c-Jun induction in the pancreas will help explain themay contribute to an increased incidence of non-healing wounds. Nerve role of this organ in multiple organ failure after burn injury.growth factor (NGF) has been reported to augment wound closure.We hypothesized that topical 2.5S NGF, a biologically active subunit

114.of NGF-b (7S NGF), accelerates wound repair and increases IN VIVO TISSUE REMODELING IN HUMAN SKIN CONSTRUCTinflammation in excisional wounds in obese diabetic mutant mice. IMPLANTS.

A full thickness 6mm punch biopsy was created on the dorsum of J Hardin-Young1, J Teumer1, L Xu1, K Kriwet1, Sylvianne Guerret2,Emmanuel Govignon1, Vincent Ronfard1 and Daniel Hartmann2.C57BL/6J-m � Leprdb mice (db/db) and heterozygous (db/-) littermates

(The Jackson Laboratory) and covered with a semi-occlusive dressing. 1Organogenesis Inc., Canton MA, USA. 2Novotec, 8 rue HermannFrenkel, Lyon-France.Mice received daily application of 50ul normal saline (NS) or 2.5S NGF

on post injury days 0-6 by injection under the dressing onto the woundGraftskin, a bilayered skin substitute, combines epidermal and dermalbed. Experimental groups (6 mice in each) included: 1) db/db mice

treated with NS, 2) db/db mice treated with NGF (1ug/d), 3) db/db substitutes in a tissue-like construct that closely resembles normal skinin form and function. Graftskin is FDA approved for treatment ofmice treated with NGF (10ug/d), 4) db/- mice treated with NS, 5) db/-

mice treated with NGF (1ug/d), 6) db/- mice treated with NGF (10ug/ diabetic foot and venous leg ulcers, but the mechanism for its efficacyis not fully understood. This study was designed to determine thed). Wounds were serially photographed and % wound epithelialization

and contraction were measured using Adobe Photoshop�. Wounds physical properties of graftskin that are important to in-vivoacceleration of healing, and to investigate the long-term changes thatwere harvested for H&E analysis at the day of closure. Healing time,

wound epithelialization, contraction and degree of inflammation were occur in-vivo to both the matrix and cellular components.compared using a Student’s t-test.

In this study, full-thickness excision wounds were created on the backof athymic mice. A piece of graftskin was placed onto each wound.Healing times in the db/db mice decreased from 29.9d in mice treated

with NS to 25.8d in mice treated with 1ug/d 2.5S NGF (p < 0.05) and The wounds were excised and assayed for histological,immunohistochemistry and electron microscopy.24.3d in mice treated with 10ug/d 2.5S NGF (p < 0.005). This trend also

occurred in the db/- mice but was not significant. These data contrastGraftskin was found to integrate rapidly with mouse tissue (7 days).with a parallel study that demonstrated that topical application of 7S

NGF results in no significant difference in time to closure compared Epidermal maturation and functional integrity were rapidly established.The basement membrane that is already formed in-vitro allowed for ato NS treated wounds. We also show that treatment with both

concentrations of 2.5S NGF results in less wound contraction and more rapid establishment of complete basement membrane structurewithin 7 days after grafting. After 14 days, the values for percutaneousimproved epithelialization in the db/db mice (p < 0.005). Histologic

evaluation of healed wounds demonstrated no difference between db/ absorption were in the range of values that are know for human normal.Bovine type I collagen was progressively replaced by human and mousedb treatment groups using a semi-quantitative inflammation score.

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001168 Meeting Schedule and Abstracts

matrix components (type I collagen and elastin). Graftskin transcription factors were upregulated in relaxin-treated THP-1 cells.Cathepsin B, a cysteine protease, showed the largest differentialkeratinocytes and fibroblasts persisted for at least one year after

grafting. expression in rhRlx-treated cells, increasing nearly 2-fold after 8-hrstimulation and more than 6-fold after 24-hr stimulation. This result

Graftskin, containing living normal cells, functions similarly to human was confirmed using quantitative real-time Polymerase Chain Reaction(PCR). Furthermore, increased Cathepsin B protein expression in THP-skin when grafted onto mice in regard to establishment of basement

membrane and barrier function. The original bovine matrix is 1 cells treated with relaxin for 24 hours was detected by Western blotusing specific anti-human Cathepsin B antibodies. Because Cathepsinprogressively replaced with newly synthesized human and mouse

matrix components. B has been implicated in several aspects of wound healing, includingmacrophage migration, extracellular matrix degradation, andangiogenesis, this finding is of potential importance in understanding115.

relaxin&#8217;s impact on wound repair.MULTIFUNCTIONAL ACTION OF RELAXIN IN WOUND HEALINGSTIMULATION.

117.Shu-Hui Liu, Usha Deshpande, Qiang Zhang, Elaine Unemori and Mark PHARMACOLOGICAL ACTIONS OF RECOMBINANT HUMANErikson. Connetics Corporation, Palo Alto, CA. RELAXIN (RHRLX) IN IMPAIRED WOUND HEALING I: ENHANCES

MACROPHAGE INFILTRATION IN DB/DB MICE.Relaxin, a naturally occurring reproductive hormone known to promoteangiogenesis and tissue remodeling during pregnancy, has been shown G. Arnold, L. Guzman, Q. Zhang, E. Unemori, and X. Huang. Connetics

Corp., Palo Alto, CA.to stimulate ischemic wound healing in animal models. To dissect themolecular mechanisms of relaxin-assisted wound healing, we have

Relaxin, a reproductive hormone, has several biologic actions includingidentified potential cellular targets of relaxin to uncover thedownstream pathways following relaxin stimulation. A human the ability to promote vasodilation and angiogenesis. The purpose of

this study was to evaluate the effect of systemic administration ofmonocyte/macrophage cell line, THP-1 was previously shown tocontain high-affinity relaxin binding sites and increased expression of recombinant human relaxin (rhRlx) on the healing of impaired wounds

using the db/db mouse model. Full thickness excisional wounds (1.5vascular endothelial growth factor (VEGF) in response to relaxinstimulation. VEGF is a potent mitogen that promotes angiogenesis and cm � 1.5 cm) were made in the back region of db/db mice. RhRlx

was delivered systemically via an Alzet� pump implanted at the timevasodilation by upregulating nitric oxide and prostacyclin productionin endothelial cells. Further analysis of the conditioned media derived of surgery. Wound size was monitored over the course of the study.

Mice treated with rhRlx had greater wound closure than did controls.from 24 hour relaxin-treated THP-1 cells also showed elevated levelsof cytokine interleukin-8 (IL-8) and urokinase type plasminogen Wound area on Day 14 was as follows (expressed as % of original area):

vehicle 91.4%, rhRlx 64.0%, p < 0.05. Histological evaluation of tissueactivator (uPA). IL-8 mediates cellular inflammatory and angiogenicresponses, and uPA plays a pivotal role in cell migration during wound collected from the wound area on Day 14 revealed increased

vasculature and increased granulation tissue formation.repair, through its activity in regulating the reorganization ofextracellular matrix. These observations are consistent with the Immunohistochemical staining using a monoclonal antibody to

macrophages revealed an increased macrophage population in thehypothesis that relaxin can enhance the angiogenic and tissueremodeling effects of macrophages at the wound sites. In addition, we rhRlx treated animals compared to vehicle treated animals. Stained

tissues were examined under 40x magnification and a field near thehave identified a human coronary artery smooth muscle cell (CASMC)that binds radiolabeled relaxin and exhibits dose-dependent cAMP edge of the wound area was scored for the number of macrophages

present on a scale ranging from 0 to 4. Mean scores were: vehicle 1.67,responses. Upon treatment with relaxin, CASMC also showed elevatedsecretion of VEGF into the culture media, under both normal and rhRlx (0.1 mg/kg/day) 2.5, rhRlx (1.0 mg/kg/day) 3.5. These data suggest

that rhRlx stimulates a macrophage response resulting in an increasedhypoxic conditions. VEGF upregulation in smooth muscle cells byrelaxin could have direct effects on the adjacent endothelium leading number of macrophages in the wound area, which in turn increases

angiogenesis and granulation tissue formation to promote woundto increased vascular permeability, endothelial cell proliferation andangiogenesis. Taken together, these results indicate that, by modulating healing.the activity of a variety of cell types to promote angiogenesis, tissueremodeling and vasodilation, relaxin plays a multifunctional role to 118.

facilitate the wound healing process. THE GENETICALLY DIABETIC MOUSE AS A MODEL TO STUDYWOUND HEALING.

116.SR Sullivan, RA Underwood, MA Antezana, ML Usui, NS Gibran, JEMICROARRAY GENE ANALYSIS IDENTIFIES MACROPHAGEOlerud. Department of Medicine (Dermatology) University ofACTIVATION MARKERS DIFFERENTIALLY EXPRESSED IN AWashington, Seattle, WA., W Carter, PhD, Fred Hutchinson CancerRELAXIN-TREATED HUMAN MONOCYTIC CELL LINE.Research Center.

Qiang Zhang, Shu-Hui Liu, Martyn Lewis, Xinfan Huang, Mark Erikson,and Elaine Unemori. Connetics Corporation, Palo Alto, CA. The genetically diabetic db/db mouse has been extensively used as a

model of delayed wound healing for the study of exogenously appliedRelaxin, a naturally occurring hormone, plays a major role in multiple substances. Wound size, shape, number/animal, coverage and controls

varies from one study to another. The purpose of our study was tobiological events including the promotion of angiogenesis andvasodilation, and the inhibition of fibrosis. Our previous work has systematically evaluate a new model using multiple punch biopsies to

create wounds on a single mouse. Four, full thickness, 6 mm punchshown that systemic administration of recombinant human relaxin(rhRlx) can stimulate wound healing in preclinical animal models. biopsy wounds, one centimeter apart, were created on the backs of

db/db mice and their db/- control littermates. The wounds were eitherRhRlx triggered selective angiogenesis at the wound sites, potentiallythrough both vascular endothelial growth factor (VEGF) and basic covered with an adhesive Tegaderm� dressing or left open to air. The

mice were sacrificed when the wounds were determined, by grossfibroblast growth factor (bFGF), which are upregulated in wound cellsfrom rhRlx-treated animals. We have also shown that the human examination, to be closed.monocytic cell line, THP-1, may be a suitable model to study aspectsof relaxin biology, as they express high affinity relaxin receptors and Hematoxylin and eosin (H&E) stained paraffin sections of the wounds

were used to confirm epidermal closure. In addition, tests wererespond to the ligand with increased expression of VEGF and bFGF.In an effort to further understand the effects of relaxin on additional conducted to see if topically applied solutions spread from wound to

wound using blue tissue dye (Bradley Products, Inc.) and applicationgene products that may be involved in wound healing, we employedDNA microarray analysis on THP-1 cells treated with relaxin at 2, 8, of biotinylated laminin 5 (b-Lam 5) for 7 days with subsequent detection

using streptavidin CY5. Results indicated that 6 mm punch biopsiesand 24 hours. The cDNA made from purified THP-1 mRNA was labeledwith fluorescent dye and hybridized to a DNA microarray, containing close more rapidly than 1.5x1.5 cm wounds and continuous wound

coverage greatly affected wound closure time (27 days for covered vs9,182 human genes, representing 7% of the human genome. Severaltypes of novel genes including proteases, cytokines, kinases, and 13 days for uncovered). This effect is likely due to inhibition of wound

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 169contraction by the dressing. Neither the topically applied b-Lam 5 nor 121.

ACUTE PRODUCTION OF LEPTIN IN EXPERIMENTAL WOUNDS.the blue tissue marking dye spread to other wounds on the mice. Thefour 6 mm punch biopsy model 1) reduces time to wound closure M R. Sierra-Honigmann1, A.K. Nath2, G. Ambrosini3 and J. Flores-studies from 50 days to 27 days 2) permits use of both treatment and Riveros3. 1Department of Plastic Surgery University of Southerncontrol wounds on the same animal, and 3) decreases the number of California, Cedars Sinai Medical Center, Los Angeles CA; 2Yaleanimals needed for each study. University School of Medicine, New Haven CT and 3Institute for

Diabetes Discovery, Branford CT.119.

Recent reports by our laboratory and others have demonstrated thatSPARC-NULL MICE EXHIBIT ACCELERATED WOUND HEALING,exogenous leptin enhances wound healing and reverses the healingALTERED DERMAL EXTRACELLULAR MATRIX, AND INCREASEDimpairment of leptin-deficient mice. We also reported that high levelsCELLULAR INVASION IN A SPONGE MODEL OF ANGIOGENESIS.of leptin are present in wounds. We performed in situ hybridizationexperiments in frozen sections of experimental wounds to address theAmy D. Bradshaw, May J. Reed, E. Helene Sage. The Hope Heartquestion of whether the leptin found in wounds resulted fromInstitute, Seattle WA and University of Washington, Seattle WA.recruitment of circulating leptin into the site or by increased productionof leptin in the wound milieu. We found an early increase in leptinSPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-mRNA levels in the basal keratinocyte layer and in dermal fibroblasts40) has been characterized in vitro as an inhibitor of cell proliferationadjacent to the wound. We also examined the production of leptin inand as a counter-adhesive factor. In addition, SPARC has been shownvitro using cultured dermal fibroblasts exposed to conditions thatto a) bind to and/or diminish the activity of certain growth factors, b)mimic the wound environment, such as hypoxia. Leptin productionassociate with ECM components including collagens, c) increase matrixwas measured by radioimmunoassay of culture medium and bymetalloprotease activity, and d) modulate the expression of specificNorthern blot analysis. We found that, when exposed to hypoxia,proteins implicated in the turnover of ECM. Expression of SPARC isfibroblasts are induced to produce leptin. We also evaluated the levelsassociated with events that involve matrix degradation and subsequentof circulating leptin after wounding and found a 6-fold peak (from 2synthesis of new matrix. Given the collagenous milieu of the dermis,to 12 ng/ml), by 12 hours, returning to basal levels by 24 hours. Tothe skin provides an excellent tissue to analyze the function of SPARCdetermine if the peak of circulating leptin originates from the woundin vivo. Preliminary observations suggest that the skin of SPARC-nullsite, we wounded human skin grafts of human-SCID mouse chimeras.mice has reduced tensile strength and that the collagen fibrils in theWe found that human leptin appeared in the mouse circulation afterdermis are smaller and more uniform in diameter than those of wild-wounding. We concluded that leptin is acutely produced in woundstype mice. We present evidence that, in the absence of SPARC, healingand that changes in circulating leptin observed several hours afterof excisional wounds is accelerated in mouse skin. An increase in thewounding originate from the wound site.percentage of proliferating cells was not found in SPARC-null versus

wild-type wounds. However, the capacity of SPARC-null dermal122.fibroblasts to migrate in vitro in response to a wounded monolayer is

IS ANTIMICROBIAL EFFICACY SUFFICIENT? A QUESTIONincreased in comparison to wild-type cells. We also observe an increaseCONCERNING THE BENEFITS OF NEW DRESSINGS.in the cellular invasion and vascularization of polyvinyl alcohol sponges

implanted subcutaneously in SPARC-null versus wild-type animals. Kan Lam, J. Barry Wright, and Robert E. Burrell. Westaim BiomedicalThese data provide evidence for a function of SPARC in the Corp., Fort Saskatchewan, AB.maintenance of connective tissue integrity and in response to injury.Elucidation of the mechanisms by which SPARC influences cell Clinical research and general experience is accentuating thebehavior will provide valuable insight into the biology of wound healing. importance of minimizing the bacterial load in both chronic and acute

wounds. This understanding, combined with the increasing prevalenceof antibiotic resistant bacteria in both the hospital and community120.setting, is driving the development of new antimicrobial dressings.ACTIVE WARMING FOR HAND ISCHEMIA IN A PATIENT WITHVarious products are touting their antimicrobial efficacy but little isARTERIOVENOUS FISTULA (STEAL SYNDROME).described relative to their impact on wound healing. We examined twonew dressing products, a nanocrystalline silver-coated antimicrobialJM West, N Puzziferri, L Reilly, TK Hunt and HW Hopf. University ofdressings (SCD) and a gauze dressing impregnated withCalifornia, San Francisco, CA 94143-0522.polyhexamethylene biguanide (PHMB), in both in vitro tests and ananimal model of delayed wound healing. Both dressings wereIschemia of the hand may occur after arteriovenous access graft surgerydemonstrated to have potent bactericidal effects in vitro when bacteriain patients with severe peripheral arterial disease. Fingertip necrosiswere applied directly to the dressing. However, the PHMB dressingand pain can result. A 53 year old man with long-standing insulin-did not have any activity beyond its own borders whereas the SCDdependent diabetes developed progressive, painful recurrent necrosisdemonstrating antimicrobial activity that diffused into the surroundingof three fingertips after an arteriovenous (AV) shunt was placed forarea, as shown in zone of inhibition assays. In the wound healingrenal dialysis. His fingers were chronically cold and cyanotic. A radialexperiments, wounds dressed with the SCD dressing developed a fullpulse was present. Palmar fasciectomy and digital sympathectomy hadgranulation bed much faster than those dressed with the PHMB dressinglost effect after two years. We hypothesized that excess sympatheticand also demonstrated a lower wound bacterial load than those dressedreactivity, fluid volume changes after dialysis, and chronic arterialwith the PHMB dressing. Over the first several days of the experimentdisease contributed to tissue hypoxia and progressive tissue loss. Wethe PHMB-dressed wounds continued to bleed to greater extent thanproposed a course of active warming to decrease vasomotor tone andthe SCD-dressed wounds. These results suggest that in clinical use,increase oxygenation. Baseline peri-wound transcutaneous oximetrysimply being an effective antimicrobial is not sufficient to make a(PtcO2, Novametrix, Wallingford, CT), was < 10mmHg and minimallyproduct the best choice to promote wound healing. This is consistentresponsive to supplemental oxygen. The patient was treated 18 hourswith results obtained with other topical antimicrobial products thata day over a six month period with active Warm-Up wound therapymay be effective against the contaminating organisms but which maysystem (Augustine Medical Inc., Eden Prairie, MN). Pain and patientimpede wound healing.comfort were monitored by visual analog scale (VAS). PtcO2

measurements were repeated after 8 weeks of active warming. Warmingat 38�C increased PtcO2 values to > 30mmHg in the hand, improvedresponsiveness to supplemental oxygen, and allowed completeseparation of necrotic tissue without infection and without need forsurgical debridement. Pain was reduced to VAS < 3 and is now absentfor hours at a time. Local active warming appears to have overcomevasoconstriction induced by the AV fistula, pain, hypovolemia, andambient cold. The patient has elected to continue his therapy severalhours a day. Supported by NIH GM27345.

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001170 Meeting Schedule and Abstracts

Although the literature suggests that values below 20 mmHg are not123.

SPLIT THICKNESS SKIN GRAFT FIXATION WITH FIBRIN SEALANT compatable with healing , 64% (164/256) of patients were improvedafter HBO2T despite a SLair TCOM < 20mmHg. Patients with an airSPRAY.value > 25 mmHg are 2.5 times more likely to benefit from HBO2T

M Spies*, M Thurnher#, LD Traber*, DN Herndon*, HR Redl#. than those with a TCOM < 25 mmHg (p � 0.001). However, this testShriners Burns Hospital Galveston, Texas, USA* and Ludwig- was only 54% reliable at predicting outcome. If the baseline TCOMBoltzmann-Institute for Experimental and Clinical Traumatology (SLair) was more than 25 mmHg, AND the transcutaneous valuesVienna, Austria#. increased by more than 20 mmHg with SLO2, the failure rate of HBO2T

was only 14% (23/136), and this was 76% reliable. For those who didEarly and stable coverage of large wound surfaces is of major not meet this criteria the failure rate was 40.7% (43/118). Only 18% ofimportance in the treatment of severe burns. Fibrin sealant (Tisseel�) patients with an in-chamber TCOM < 100 mmHg were helped (2/11).has been shown to improve hemostasis on wound surfaces and promote Of those with in-chamber TCOM levels of > 200 mmHg, 78% werewound healing. A newly FDA approved spray system (Tissomat�) helped. This test was also 76% reliable in predicting outcome. Althoughallows coverage of extensive wound surfaces with a thin fibrin film. there may be some variability in data collected from different facilities,A thrombin component of 500 IU/ml (FS500) results in a fast and a 4 the quality of these measurements probably correlates well with generalIU/ml (FS4) in a slow setting rate. use.

We propose that the use of Tisseel is an efficient approach to achieve These data confirm prior observations that while sea level air TCOMfixation and an excellent take rate of autologous sheet skin grafts may be a good way to determine which patients will not healcomparable to staples or sutures, but with definite advantages in spontaneously, it is not a good way to determine which patients willhandling. benefit from HBO2T. The best predictor of success with HBO2T is an

in-chamber value > 200 mmHg. Alternatively, a baseline TCOM > 25Full-thickness excisional wounds (5x8cm), (8/group) were covered followed by an increase with SLO2 of > 20 mmHg is 76% reliable. Thuswith split thickness sheet skin grafts (10/1000’’) and randomly either the reliability of TCOM alone in predicting benefit from HBO2T is, atfixed with sutures (SG), fibrin sealant with 500 IU/ml (FS500), or 4 IU/ best, 78%.ml (FS4) thrombin component in a pig model. Assessed was ease andspeed of application during the procedure and take rate. Complications, 125.such as dislocation or hematoma formation were documented over a THE POTENTIAL FOR RAPID HEALING OF VENOUS LEG ULERS3 week period by digital imaging. Statistics: ANOVA/ Tukey‘s pairwise WITH THE SIMULTANEOUS USE OF BIOENGINEERED HUMANcomparison. Data as mean � STD. SKIN EQUIVALENT (APLIGRAF) AND SUBFASCIAL PERFORATOR

SURGERY.The application time was shortened by fibrin sealant (FS4 4.4 � 0.6

Thomas Serena, Kimberly Campbell and Carrie Corbran. Penn Northmin, FS500 3.8 � 0.9 min, SG 16.4 � 3.1 min, p < .01) and ease ofCenter For Advanced Wound Care, Warren, PA. and Gannon University,usage was best with FS4 for its slower setting time, allowing forErie, PA.corrections. At day 5 in four wounds partial dislocation of the skin

graft occurred with FS500 relating to19.2 � 26.7% of the initial woundVenous leg Ulcers affect two and one-half million individuals in thearea, compared to one each in FS4 (1.6 � 4.0%) and SG (1.0 � 2.2%)United States. Most of these ulcers are healed with nonoperativerespectively. When accounting for additional hematoma formationmeasures such as compression, extremity elevation and patientunder the graft, this is a 5 day take rate of 94 � 6% for FS4 (p < .05education. However not all patients achieve healing and recurrencevs. FS500), 64 � 22% for FS500, and 91 � 11% for the SG (p < .05 vsrates are high. Both bioengineered human skin equivalent (Apligraf)FS500) and at day 14 - 94 � 11% for FS4 (p < .05 vs. FS500), 58 �and subfacial endoscopic perforator surgery (SEPS) have been shown21% for FS500, and 98 � 3% for SG (p < .05 vs FS500). At day 21 98to be effective in healing venous leg ulcers.� 5% of the wound area in the FS4, 83 � 16% in the FS500, and 95

� 14% in the SG group was healed.We report four cases of hard-to-heal venous ulcers (duration greaterthan one year) in which a SEPS procedure was performed inThe fixation of skin sheet grafts can be improved and eased by theconjunction with the application of bioengineered skin (Apligraf) touse of fibrin sealant spray. The slow setting fibrin sealant containingthe active ulcer. All the patients had longstanding venous ulcerations4IU/ml thrombin was easier to use compared to the fast 500 IU/ml and(range 1-10 years). They all had lipodermosclerosis and activeshowed better macroscopic outcome results.ulceration (class C6). One patient had two venous ulcers. All had failednonoperative therapy. Venous duplex studies revealed large

124. incompetent perforating veins in all four patients. SEPS was performedTHE PREDICTIVE VALUE OF TRANSCUTANEOUS OXYGEN and an average of ten perforating veins per patient divided. TheTESTING IN SELECTING HYPERBARIC OXYGEN PATIENTS: bioengineered skin (Apligraf) was fenestrated and applied to theRESULTS OF A LARGE RETROSPECTIVE STUDY. ulceration. The leg was then treated with elastic compression.

CE Fife, C Buyukcakir,G Otto, P Sheffield, J Mader, R Warriner. Dept. Total operative time averaged less than sixty minutes. Of the five ulcers,of Anesthesiology, The University of Texas Medical School at Houston, two healed within two weeks. Both of these patients returned to workand the Hermann Center for Wound Healing, Houston,TX, College of within a month of surgery. The third patient, who had two ulcers,Business, Univ. of North Carolina at Charlotte, Charlotte, NC, The Nix healed the first ulcer within three weeks; however, the second ulcerHyperbaric facility, San Antonio, TX, Dept. of Internal Medicine, The was not completely healed until twenty weeks postoperatively. In theUniversity of Texas Medical Branch at Galveston, TX, Southeast Texas fourth patient the ulcer was 90% healed within twelve weeks but didCenter for Wound Care and Hyperbaric Medicine, Conroe, TX. not achieve complete healing for twenty-eight weeks. At nine months

follow-up, one patient had developed a recurrence, which rapidlyAlthough widely used for patient selection, transcutaneous oximetry healed with compression alone.(TCOM) is not a sensitive test for outcome prediction. Small studieshave suggested that sea level or in-chamber oxygen administration The use of bioengineered human skin equivalent (Apligraf) inimproves the predictive value of TCOM in selecting patients likely to conjunction with the SEPS procedure may be helpful in dealing withbenefit from hyperbaric oxygen therapy (HBO2T). the hard-to-heal venous stasis leg ulcer.

Five hyperbaric facilities supplied retrospective data on 1006 diabeticpatients who underwent HBO2T for wound healing and whose outcomewas described as healed, partially healed, not improved, or amputated.TCOM values, measured next to the wound, were recorded under threeconditions: breathing air at sea level (SLair), breathing oxygen at sealevel (SLO2), and breathing oxygen in the hyperbaric chamber.

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 171

126. and shorter hospital stays. The combination of irrigation and negativepressure therapy appears to be effective in treating chronic woundsINTEGRATED PAIN MANAGEMENT AS PART OF THE TREATMENT

OF CHRONIC WOUNDS. infected with antibiotic resistant bacteria.

Freedman G*, Brem H, Kapil-Pair N, Kaplan D, Hollier L. Mount Sinai128.Medical Center, Department of Anesthesiology*, Department of

IMPROVING ACCURACY AND SENSITIVITY OF BLOOD PERFUSIONSurgery, New York, NY.ASSESSMENT.

Wound pain management is as specific as the etiology of the chronic R Overby, D Feldman, D Kilpadi, S Range, and D Wirthlin1, Departmentwound itself and specific guidelines should be used to manage pain of Biomedical Engineering, Department of Surgery1, University ofin wound care patients. The physician managing pain in the wound Alabama at Birmingham, Birmingham, AL.patient and the wound care doctor meet on a weekly basis to examineand review the total and comprehensive management of the patient. To accurately assess the effectiveness of wound treatments,We have based these guidelines after examining multiple consecutive quantitative assessments are essential; which is hampered by thepatients as a team. The clinicians must have all pain options as part current lack of validated quantitative clinical measures. It is alsoof their armamentarium. In patients with arterial insufficiency, painful important to use these measures to determine a tissue state, relativeulcers secondary to arteriosclerosis is best treated by sympathetic to the normal state, as well as track this tissue state during treatment.blockade to the area of the ulcer. In patients with venous reflux ulcers, Therefore, interventions can be assessed on the ability to move theanalgesic regimens frequently used for these patients rely on oral tissue state toward the normal state. The difficulties, however, are thechronic pain medications. One regimen includes a long acting/ lack of consensus on parameters to characterize the normal tissuecontinuous release opioid as a pill or a patch, to maintain analgesic state, the lack of validated quantitative measures for assessing theselevels of the opioid consistently in the therapeutic window, giving better parameters, and the relationship between these parameters and clinicalanalgesia with fewer side effects. A nonsteroidal anti-inflammatory outcome. For skin wounds, tissue state assessments can be done indrug and a tricyclic antidepressant frequently are employed for their three groups: healing rate, tissue health, and tissue function. Healinganalgesic effects. A fast onset, short duration opioid is utilized for rate is the speed in which the wound is closed, independent of woundepisodic breakthrough pain. In patients with diabetic neuropathic size. Tissue health is an assessment of the wound microenvironmentulcers, analgesia is best facilitated with anticonvulsants, tricyclic (e.g. blood flow or oxygen levels) in an effort to determine the abilityantidepressants and possibly with N-Methyl D-Aspartate receptor of the wound to heal. Tissue function is an assessment of the skin’sinhibitors or alpha-2 agonists. In patients with decubitus ulcers, chronic ability to perform its normal functions (e.g. mechanical properties ororal analgesic regimens and neuropathic medications are frequently epidermal barrier functions). Ultimately the ability to predict clinicalused. In all chronic wounds, the most important part of the pain regimen outcome needs to be determined for these tissue state measures.is removing local infection. A strong understanding of thepathophysiology of the pain and all the treatment options available are The goal of the current study, is to determine the utility of bloodessential for effective analgesia in this patient population. Daily and perfusion (Scanning Laser Doppler—-SLD) for skin ulcers. This wasweekly discussion of the pain and wound management can result in done in three parts: 1) To determine parameters, which affect thedecreased length of stay and significant decrease in patient morbidity. quantitative nature of the SLD, 2) to examine the temporal and spatial

relationships of the SLD, and 3) to compare to TcPO2/TcPCO2. It was127.found that provocations (heating, inspired oxygen, and limb elevation)STERILIZING CHRONIC WOUNDS WITH NEGATIVE PRESSUREwere useful in increasing the sensitivity of the SLD, with heat beingTHERAPY: THE ROLE OF ANTIBIOTIC IRRIGATION.the most effective provocation. The SLD measure at the site of the

Subhas C Gupta. Division of Plastic Surgery, Loma Linda University, heated O2 probe appears to correlate well with the tissue gas measures.Loma Linda, CA. The SLD, however, has the added advantages of spatial resolution and

the ability to measure the wound directly as well as adjacent to it. InWound management is an ever-expanding concern for health care ongoing studies, the ability of the SLD to predict clinical outcome isproviders, insurance companies, and the government. Treating chronic being evaluated.wounds has been estimated to cost between $5 and $7 billion annually,and these wounds are increasing at a rate of 10% per year. Additionally, This study was supported by CDC-NCIPC, UAB ICRC, and UAB HSF.the rate of antibiotic-resistant invasive wound infections in chronicwounds is growing rapidly. This is compounding the cost issue, and

129.prolonging duration of care of patients. There remains no effective STRESS AND COPING RESPONSES INFLUENCE WOUND HEALING.means of eradicating resistant organisms from chronic wounds.

Joie Whitney, Suzanne Clark, Stacy Heiner, Monica Jarrett, MargaretHeitkemper, Lori Loan. University of Washington, Seattle, WA. andSince its introduction in 1995, the VAC has been used to treat a variety

of wound types with success. This study will evaluate the ability of Madigan Army Medical Center, Tacoma, WA.the VAC system to sterilize chronic wounds. We have developed anantibiotic delivery system that works in conjunction with the VAC, and The cumulative effects of psychological, physical and surgical stress

result in hypothalamic-pituitary-adrenal (HPA) mediated peripheralhave preliminary data from our pilot study demonstrating clearanceof oxacillin resistant Staphylococcus Aureus from chronic wounds. We vasoconstriction and elevated serum cortisol levels, both of which have

been implicated in wound healing. The purpose of this study was towill determine the value of using antibiotic irrigation in conjunctionwith the VAC for the purpose of sterilizing wounds. describe the effects of preoperative psychological stress and

postoperative physiological stress responses on wound healing to gainWe have planned a prospective randomized double blind trial to insight into individual characteristics most likely to equate with

efficient wound healing.evaluate our hypothesis. Patients with open wounds of greater thanone month’s duration with quantitative wound cultures indicating

Method. Seventy-seven subjects (mean age 47 � 20) undergoing kneebacterial counts > 105 /gram of tissue will be recruited for the study.All patients successfully recruited into the study will be evaluated pre- arthroscopy or arthroplasty participated. Subjects completed

questionnaires capturing preoperative psychological stress and coping, intra-, and post-treatment. The data and digital imaging will berecorded in a multimedia database that will allow for assessment of strategies. Postoperative urine samples (days 0-7) were analyzed for

catecholamines and cortisol. Wound healing (histology,multiple outcome parameters.hydroxyproline, pro alpha 1[I] collage mRNA) was evaluated in tissueof subcutaneous polytetrafluoroethylene (ePTFE) implants removedThe following measures were studied over time: wound size,

dimensional volume, tracking, drainage, granulation tissue, quantitative on the 7th postoperative day.cultures, length of stay, and delay to surgical closure.

Results. Hydroxyproline accumulation was inversely related toepinephrine levels on postoperative days 1 and 2 (-.304,p � .008; -.262,Both saline irrigation and antibiotic irrigation significantly reduced the

bacterial load in the chronic wounds. This led to earlier surgical closure, p � .02) and norepinephrine levels on postoperative day 2 (-.250, p

WOUND REPAIR AND REGENERATIONMARCH–APRIL 2001172 Meeting Schedule and Abstracts

� .03). Several specific coping approaches were associated with cytotoxicity when compared to Betadine. Additionally, our animalstudies in rabbit eyes (Draize test), demonstrated that our solution hassignificantly higher levels of hydroxproline. Other wound healing

measures were not related to psychologic or physiologic stress a safety profile comparable to saline (negative control) with noirritation or side effects, as opposed to Betadine. Furthermore, ourvariables.recent exploratory clinical studies on human wounds have confirmedthe safety of our solution as a topical wound treatment product. InConclusions. These data suggest that the ways in which individuals

prepare for and cope with the stress of pending surgery, as well as conclusion, our research demonstrates that this solution is atherapeutically efficacious antimicrobial product with markedlypost surgical HPA mediated stress have consequences for wound

healing. reduced cytotoxicity.

132.130.

EFFICACY AND APPLICATION GUIDELINES OF GRAFTSKIN FORDIABETIC NEUROPATHIC PLANTAR FOOT ULCERS.

L. Lindlsey, E. Hornby, K. Marshall. Novartis PharmaceuticalsCorporation, East Hanover, NJ.

Approximately 16 million individuals in the United States have diabetes.Diabetic foot ulcers affect 15% to 20% of this population and accountfor 86,000 amputations per year. The cost associated with this conditionexceeds $ 1 billion per year. Newer treatment modalities includinggrowth factors and tissue engineered skin, when combined with properwound care, have shown improvements in outcomes. The primaryobjective was to confirm efficacy, safety and application guidelines ofGraftskin, in patients with diabetic neuropathic foot ulcers.

This was and open-label, multi-center study of 13 adult patients withdiabetes with plantar neuropathic ulcers. The ulcers had to be presentfor at least 4 weeks prior to study participation, thus demonstratingunresponsiveness to conventional therapy. The primary efficacyvariable was time to 100% wound healing. Healing was defined as fullepithelialization of the wound with the absence of drainage.

During the 12-week treatment period up to 3 applications of Grafskincould be applied to the target ulcer with a minimum of 2 weeks betweenapplications. All patients received good wound care including surgicaldebridement, off-loading of the ulcer, and infection evaluation. Allpatients had to continue with acceptable off-loading and standardwound care procedures until the follow-up safety visit.

Graftskin achieved complete healing in 9 of 13 (69%) within the 12weeks of the study. Seven of these patients (54%) achieved completehealing after 7 weeks. An average of 1.5 applications were required toachieve complete healing.

133.There were no adverse events at the treated ulcer sites and none ofthe reported adverse events were suspected to be related to Graftskin.

This open study supports the pivotal data approved by the FDA forthe treatment of diabetic neuropathic foot ulcers. Graftskin is safe andeffective in the treatment of diabetic neuropathic foot ulcers. Additionalstudies need to be performed to substantiate application guidelines.

131.

NOVEL ANTISEPTIC, NON-CYTOTOXIC SOLUTION: POTENTIALREPLACEMENT FOR SALINE IN WOUND CARE.

Suzanne Bernard, Ron Najafi and Mansour Bassiri. NovaCalPharmaceuticals, Novato, CA.

Despite the use of antibiotics and improved sterile techniques, infectionremains to be one of the most frequent complications of wound healing.Infection must be avoided to permit proper wound healing. In thisregard, antibiotics and topical antiseptics with broad antimicrobialspectra have been used. Although antiseptics have not been reportedto develop bacterial resistance like antibiotics, several reports havesuggested that antiseptics exhibit significant cytotoxicity. Such effectsmay interfere with wound healing process. The purpose of this studywas to investigate a topical anti-microbial saline solution, to controlinfection in wound healing. In our experimental design, we comparedthe bactericidal efficacy of our solution with some commonly usedantiseptics, against a broad spectrum of microorganisms. We alsocompared its safety with the antiseptic Betadine at the sameconcentrations and exposure time intervals, using human cell cultures:fibroblasts and keratinocytes. We now have evidence that our solutionis an effective antibacterial product with significantly diminished

WOUND REPAIR AND REGENERATIONVOL. 9, NO. 2 Meeting Schedule and Abstracts 173

134. 136.

MALP-2, A MACROPHAGE ACTIVATING LIPOPEPTIDEACCELERATING WOUND HEALING.

Ursula Deiters1, Johannes Barsig2, Michael Morr1, and Peter F.Muhlradt1, 1 Gesellschaft fur Biotechnologische Forschung,Braunschweig, Germany, 2 Byk Gulden Lomberg Chemische Fabrik,Konstanz, Germany

The macrophage activating lipopeptide of 2 kDa (MALP-2) wasoriginally isolated from Mycoplasma fermentans and discovered onaccount of its macrophage stimulating activity. It causes leukocyteinfiltration, because of its capacity to induce the release of chemokines.MALP-2 may thus contribute to accelerated wound healing by attractingleukocytes to clean the wound and fight infection, and by providingactivated macrophages as a source of growth factors. The ability ofMALP-2 to attract macrophages was investigated histologically afteri.c. injection of MALP-2 in the dorsal skin of NMRI and C57BLKS/Jmice. Three days after application of MALP-2 there was a significantaccumulation of leukocytes at the injection site. Six days later newvessels appeared. No such effects were observed with vehicle controls.The half life of MALP-2 in the skin was estimated by determination ofremaining macrophage stimulating activity from skin biopsies in a nitricoxide release assay. MALP-2 activity at the site of injection decreasedrapidly but was still detectable for at least three days. In a full thicknessskin excision model the acceleration of wound closure by MALP-2 wasmeasured in healing-impaired diabetic C57BLKS/J-m�/�Leprdb mice.In this model the most effective dose of MALP-2 was determined bymeasuring infiltration and proliferation of cells by determination ofnucleic acid, and specifically the fibroblast influx by measuringhydroxyproline as an indicator of collagen synthesis. Treatment ofwounds with 5 ig MALP-2 led to maximal infiltration of cells andmaximal hydroxyproline synthesis, whereas higher MALP-2concentrations were uneffective or detrimental, respectively. Repeatedtreatment of MALP-2 significantly enhanced wound closure ascompared to vehicle control. These results show that MALP-2, a well-defined, synthetically accessible compound, accelerates wound healingby its ability to activate macrophages and possibly other cells to releasechemokines and growth factors. In this way, local infiltration of135.leukocytes is induced, which in turn promotes the formation ofgranulation tissue. It may be possible to use MALP-2 to preventinfections by pretreating surgical sites with this compound.