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S80 Surgical Forum Abstracts J Am Coll Surg

ocal reactive oxygen species scavenging improvesiabetic wound healingenis Knobel MD, James Lee Crawford BS, Parag Butala MD,dward Henry Davidson MBBS, Meredith Wetterau MD,teven Mark Sultan BA, Caroline Szpalski MD,tephen M Warren MD, FACS, Pierre B Saadeh MD, FACSew York Medical Center, New York, NY

NTRODUCTION: Hypoxia and hyperglycemia increase cellular stressn diabetes and this stress is defined, in part, by elevated reactive oxygenpecies (ROS). Although the relationship between apoptotic mediatorsnd ROS is increasingly being uncovered in other disease states, there haseen little focus on this relationship in diabetic wound healing. Weypothesize that reducing ROS with N-acetylcysteine (NAC), an ROScavenger, mitigates molecular derangements associated with diabeticounds and augments wound healing.

ETHODS: Paired 6mm stented wounds were created on diabeticice. NAC (5-600mM) in agarose-gel matrix was applied on post-ound day 1 and every other day thereafter until animals were

acrificed at day 10. Wound closure time was photometrically as-essed. Wounds were harvested for histology, immunohistochemistryapoptotic mediators, ROS, vaculogenic markers). Vasculogenic andxidation-state mediators were evaluated by ELISA and RT-PCR.

ESULTS: Doses of 40mM NAC consistently closed wounds byay 17�1. NAC at 600mM and 5mM had no effect on closure,ompared to controls (27�1 days). NAC at 40mM wounds im-roved tissue architecture, and decreased p53, caspase-3, and oxida-ive DNA. ELISA confirmed these findings with 43% decrease in53 and near 50% decrease in DNA damage. In 40mM NAC-treatedounds, there was greater CD31 endothelial marker staining, VEGF

evels more than doubled, and SDF-1 increased 3.5-fold. Addition-lly, 40mM NAC-treaded wounds had decrease expression of thero-oxidant gene PUMA (33% of control) and a decrease in thenti-oxidant gene GPX1 (64% of control).

ONCLUSIONS: Local reduction of ROS with NAC improves di-betic wound healing by reversing the apoptotic and vasculogenic

erangements. a

otch1 signaling regulates wound healingadhuchhanda Roy MD, PhD, Barbara Zarebczan MD,

andy J Schlosser BS, B Lynn Allen-Hoffmann PhD,erbert Chen MD, Timothy W King MD, PhDniversity of Wisconsin, Madison, WI

NTRODUCTION: Cutaneous wound healing is a major healthroblem in the US, often requiring skin allografts from donor cadav-rs. In an attempt to address problems associated with the usef potentially contaminated cadaveric skin, viability and supply,trataGraft, a second-generation living human skin substitute, wasecently evaluated in a phase I/II safety and efficacy clinical trial. Inhe study, StrataGraft skin substitutes, created from pathogen-free,ong-lived NIKS human keratinocyte progenitors, were found toerform comparable to cadaver allografts. However, the molecularechanism underlying NIKS wound healing capabilities remains

nclear. Recent studies suggest that Notch1 signaling is involved inoth the skin injury response and the dermal repair processes. Weypothesized that Notch1 signaling regulates NIKS wound healingroperties.

ETHODS: Notch1 mRNA levels from NIKS cells in monolayerulture were assessed by real-time PCR and Notch1 protein levelsere detected by Western Blot. To determine the role of Notch1

ignaling, proliferation and migration of NIKS were tested usingTT and scratch test assays, respectively, in the presence or absence

f DAPT, an inhibitor of Notch1 signaling.

ESULTS: High basal Notch1 mRNA expression was detected inIKS. It also expressed about two-fold higher Notch1 protein levelhen compared to parental primary BC-1-Ep keratinocytes. Block-

ng Notch1 activation by DAPT inhibited NIKS cellular prolifera-ion (30-40%, p�0.01) and migration compared to untreatedIKS.

ONCLUSIONS: Our results support the hypothesis that Notch1ignaling is involved in the wound healing mechanism in NIKS.argeted activation of Notch1 signaling at the wound site could

ccelerate cutaneous healing in a clinical setting.