AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK

Introduction: Several single gene disorders, including factor IX deficiency, require gene delivery strategies that would provide a stable, safe and sustainable systemic protein release. Previous techniques relying on viral or plasmid gene delivery are clinically limited by risk of insertional mutagenesis or poor efficiency due to gene silencing. Minicircle (MC) DNA represents an ideal gene delivery system that is episomal and resistant to in vivo gene silencing. MCs are engineered through removal of the bacterial backbone of plasmid DNA, preventing immunogenicity and subsequent transgene elimination. Adipose tissue flaps are ideal candidates for MC transfection due to their excellent vascularization, peripheral location and expendability. We propose MC transfection of an adipose tissue flap as a safe, reversible and clinically translatable gene delivery system. Methods: In vitro, murine adipocytes and adipose derived stem cells (ASCs) were isolated from inguinal fat pads and transfected with MC DNA encoding for luciferase or GFP via nucleofection. In vivo, naked MC DNA was injected into murine inguinal fat pads at two doses (10ug or 30ug, n=6), and compared to injection of PBS alone (n=4). for all experiments, successful transgene expression was determined using fluorescence microscopy for GFP and bioluminescent in vivo luciferase imaging (IVIS) for luciferase MCs. Results: In vitro nucleofection resulted in efficient transgene expression in both murine ASCs and adipocytes. In vivo luciferase MC DNA injection showed a localized, initial maximum luminescence of 1.81x106 and 9.44x105 photons/s/sr/cm2 at 30 and 10ug, respectively, compared to 1.02x104 photons/s/sr/cm2 in the negative control (p=0.02). Although luciferace expression declined initially, it remained localized to the fat pad and was traceable over a mid-term period of time. Conclusions: Vascularized adipose tissue flaps are particularly suitable for sustainable gene delivery approaches since they are easily accessible and expendable. We successfully show that transfection with MC DNA is a viable and safe in vivo gene delivery system that provides efficient mid-term transgene expression with minimal adverse effects. This has significant clinical implications, specifically with respect to the treatment of single gene disorders where a constant systemic release of a factor is required.

AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK AMERICAN SOCIETY OF PLASTIC SURGEONS PLASTIC & RECONSTRUCTIVE SURGERY PRS GLOBAL OPEN ASPS EDUCATION NETWORK

Backround: Protein delivery from transfected cells can induce expression of tissue inductive factors to stimulate the cellular processes required for regeneration. We established a cell-based, non viral gene-transfer method using fibroblasts to temporarily produce bFGF and VEGF165, as a form of pharmacological local preconditioning before tissue ischemia occurs. Material and Methods: The eukaryotic expression vectors harboring VEGF and bFGF cDNAs were transfected into rat primary skin fibroblasts mediated by Amaxa Nucleofector and optimized by our own laboratory protocol. To determine an improvement in ischemically challenged tissue, a genetically modified cellspool was injected into the target tissue 1 week before inducing an ischemic flap model. Gene expression and protein production in vivo and in vitro were measured by real time PCR and immunoassay (BioPlex) respectively. Clinical outcome was demonstrated by planimetric measurements. Results: Temporary protein expression of bFGF and VEGF165 in the target tissue of the ischemic flap model increased compared to controls after injection of genetically modified cells. A highly significant improvement of tissue survival was observed after the transfected cell administration. A reduction in flap necrosis by one-third or more was detected after two weeks if transfected cells were applied 1 week before ischemia. Conclusion: In our work we showed that temporary expression of bFGF and VEGF165 induces therapeutically relevant effects in the rat flap model of ischemia. Our standardized high efficiency non viral bFGF and VEGF165 transfection technology is now used in preclinical research.

1University of Luebeck, Plastic Surgery, Luebeck, Germany 2Fraunhofer Institute, Marine Biotechnology, Luebeck, Germany

Background: Pulsed electromagnetic fields (PEMF) are used as adjunctive therapy for delayed-union fractures, chronic wounds, and post-operative pain. PEMF potentiates the synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) through modulation of Ca2+ binding to calmodulin (CaM) affecting all aspects of tissue repair: reduced edema and pain, increased angiogenesis and cell proliferation. PEMF increased DNA synthesis in articular chondrocytes via the CaM-dependent NO/cGMP pathway, assessed using a CaM antagonist and NO and cGMP inhibitors. We have reported, in a double-blind placebocontrolled clinical study, that PEMF reduced post-operative pain by 300% at 5 hrs. post-op (P<0.001), possibly via CaMdependent reduction of IL-1 in the wound bed. PEMF has been shown to increase tubule formation in HUVEC cultures, and promote neovascularization in diabetic mouse wounds, via an effect on FGF-2 production. The goal of this study was to quantify the effect of PEMF on CaM-dependent cGMP in HUVECs.

Fascia lata grafts are commonly employed as support structures in plastic and reconstructive surgery. Despite this widespread application, little objective testing has been published regarding the biomechanical features of this graft particularly as related to the clinical relevance of directional orientation.

Medical University of Luebeck, Plastic Surgery (Luebeck, Germany), University of California, Plastic Surgery (San Diego, United States)

Pressure ulcers remain a major concern for healthcare facilities, from both economical and quality-of-care points of view, but with proper patient care their occurrence can be reduced. Interestingly, studies as rare as they are on this particular subject demonstrate that even though a 7 to 29 % range of incidents for pressure sores exists in the general acute care population, the incidence increases to an alarming 12 to 66 % rate for pressure sores acquired intra-operatively. Studies examining factors most likely associated with intra-operatively acquired pressure ulcers have not resulted in the development of reliable risk reduction tools adequately intervening in the surgical patient population.

Hibernoma is an uncommon benign fatty tumor that arises from the vestiges of fetal brown fat. We present a case report of a hibernoma of the back in a symptomatic 42-year-old man and describe the important clinical, histopathologic, and imaging findings. Computed tomography shows a well-defined hypodense mass with septations. Magnetic resonance imaging shows intermediate T1 and bright T2 signal of the mass and also demonstrates the characteristic marked contrast enhancement.

Background We have recently shown that the expression of nestin, a progenitor/stem cell marker protein, is localized in different mesenchymal compartments in human skin including the sweat gland stroma.Objectives As other exocrine glands are recognized sources of multipotent stem cell populations with potential for multilineage differentiation, it was our aim to isolate, expand and characterize glandular stem cells from human sweat glands.Methods Isolation of human sweat glands was based on mechanical and enzymatic digestion of axillary skin. Cultivation was performed on collagen-coated cell culture dishes and the resulting cell population was investigated at the protein and mRNA level.Results Outgrowing cells of isolated sweat glands showed a high-proliferation activity and were characterized by nestin expression in more than 80% of the cells. These sweat gland stem cells could be maintained in culture for long periods of time and showed spontaneous differentiation into cells representative of the different germ layers.Conclusions This pilot study provides the first, simple protocol for the isolation of adult human nestin-positive stem cells from the sweat gland mesenchyme, which promises to provide an easily accessible and abundantly available, autologous source of multipotent stem cells for cell-based regenerative medicine applications.