Dana Egozi

Improved vascular organization enhances functional integration of engineered skeletal muscle grafts

Severe traumatic events such as burns, and cancer therapy, often involve a significant loss of tissue, requiring surgical reconstruction by means of autologous muscle flaps. The scant availability of quality vascularized flaps and donor site morbidity often limit their use. Engineered vascularized grafts provide an alternative for this need. This work describes a first-time analysis, of the degree of in vitro vascularization and tissue organization, required to enhance the pace and efficacy of vascularized muscle graft integration in vivo. While one-day in vitro was sufficient for graft integration, a three-week culturing period, yielding semiorganized vessel structures and muscle fibers, significantly improved grafting efficacy. Implanted vessel networks were gradually replaced by host vessels, coupled with enhanced perfusion and capillary density. Upregulation of key graft angiogenic factors suggest its active role in promoting the angiogenic response. Transition from satellite cells to mature fibers was indicated by increased gene expression, increased capillary to fiber ratio, and similar morphology to normal muscle. We suggest a “relay” approach in which extended in vitro incubation, enabling the formation of a more structured vascular bed, allows for graft-host angiogenic collaboration that promotes anastomosis and vascular integration. The enhanced angiogenic response supports enhanced muscle regeneration, maturation, and integration.

An engineered muscle flap for reconstruction of large soft tissue defects

Significance Effective restoration of large soft tissue defects requires the use of tissue flaps, with viability that is largely determined by degree of vascularization. In view of the tedious transfer procedures and donor site morbidity associated with autologous flaps, this work set out to design and evaluate an engineered muscle flap featuring a robust vascular port formed from preseeded endothelial cells and host vasculature. The implanted flap was highly vascularized, well-perfused, and anastomosed with host vessels. Engineered flaps of this nature promise to circumvent the need to harvest and transfer massive tissue volumes, while avoiding the consequential complications. Large soft tissue defects involve significant tissue loss, requiring surgical reconstruction. Autologous flaps are occasionally scant, demand prolonged transfer surgery, and induce donor site morbidity. The present work set out to fabricate an engineered muscle flap bearing its own functional vascular pedicle for repair of a large soft tissue defect in mice. Full-thickness abdominal wall defect was reconstructed using this engineered vascular muscle flap. A 3D engineered tissue constructed of a porous, biodegradable polymer scaffold embedded with endothelial cells, fibroblasts, and/or myoblasts was cultured in vitro and then implanted around the femoral artery and veins before being transferred, as an axial flap, with its vascular pedicle to reconstruct a full-thickness abdominal wall defect in the same mouse. Within 1 wk of implantation, scaffolds showed extensive functional vascular density and perfusion and anastomosis with host vessels. At 1 wk posttransfer, the engineered muscle flaps were highly vascularized, were well-integrated within the surrounding tissue, and featured sufficient mechanical strength to support the abdominal viscera. Thus, the described engineered muscle flap, equipped with an autologous vascular pedicle, constitutes an effective tool for reconstruction of large defects, thereby circumventing the need for both harvesting autologous flaps and postoperative scarification.

Erythropoietin Improves the Survival of Fat Tissue after Its Transplantation in Nude Mice

Background Autologous transplanted fat has a high resorption rate, providing a clinical challenge for the means to reduce it. Erythropoietin (EPO) has non-hematopoietic targets, and we hypothesized that EPO may improve long-term fat graft survival because it has both pro-angiogenic and anti-apoptotic properties. We aimed to determine the effect of EPO on the survival of human fat tissue after its transplantation in nude mice. Methodology/Principal Findings Human fat tissue was injected subcutaneously into immunologically-compromised nude mice, and the grafts were then treated with either 20 IU or 100 IU EPO. At the end of the 15-week study period, the extent of angiogenesis, apoptosis, and histology were assessed in the fat grafts. The results were compared to vascular endothelial growth factor (VEGF)-treated and phosphate-buffered saline (PBS)-treated fat grafts. The weight and volume of the EPO-treated grafts were higher than those of the PBS-treated grafts, whose weights and volumes were not different from those of the VEGF-treated grafts. EPO treatment also increased the expression of angiogenic factors and microvascular density, and reduced inflammation and apoptosis in a dose-dependent manner in the fat grafts. Conclusions/Significance Our data suggest that stimulation of angiogenesis by a cluster of angiogenic factors and decreased fat cell apoptosis account for potential mechanisms that underlie the improved long-term survival of fat transplants following EPO treatment.

Fibronectin Potentiates Topical Erythropoietin-Induced Wound Repair in Diabetic Mice

Diabetes mellitus disrupts all phases of the wound repair cascade and leads to development of chronic wounds. We previously showed that topical erythropoietin (EPO) can promote wound repair in diabetic rats. Fibronectin (FN) has a critical role throughout the process of wound healing, yet it is deficient in wound tissues of diabetic patients. Therefore, we investigated the effect of topical treatment of both EPO and FN (EPO/FN) on wound repair in diabetic mice. Full-thickness excisional skin wounds in diabetic and nondiabetic mice were treated with a cream containing vehicle, EPO, FN, or EPO/FN. We assessed the rate of wound closure, angiogenesis, apoptosis, and expression of inflammatory cytokines, endothelial nitric oxide synthase (eNOS) and β1-integrin, in the wound tissues. We also investigated the effect of EPO, FN, and EPO/FN on human dermal microvascular endothelial cells and fibroblasts cultured on fibrin-coated plates, or in high glucose concentrations. EPO/FN treatment significantly increased the rate of wound closure and this effect was associated with increased angiogenesis, increased eNOS and β1-integrin expression, and reduced expression of inflammatory cytokines and apoptosis. Our findings show that EPO and FN have an additive effect on wound repair in diabetic mice.

Adipose-derived endothelial and mesenchymal stem cells enhance vascular network formation on three-dimensional constructs in vitro

BackgroundAdipose-derived mesenchymal stem cells (MSCs) have been gaining fame mainly due to their vast clinical potential, simple isolation methods and minimal donor site morbidity. Adipose-derived MSCs and microvascular endothelial cells have been shown to bear angiogenic and vasculogenic capabilities. We hypothesized that co-culture of human adipose-derived MSCs with human adipose-derived microvascular endothelial cells (HAMECs) will serve as an effective cell pair to induce angiogenesis and vessel-like network formation in three-dimensional scaffolds in vitro.MethodsHAMECs or human umbilical vein endothelial cells (HUVECs) were co-cultured on scaffolds with either MSCs or human neonatal dermal fibroblasts. Cells were immunofluorescently stained within the scaffolds at different time points post-seeding. Various analyses were performed to determine vessel length, complexity and degree of maturity.ResultsThe HAMEC:MSC combination yielded the most organized and complex vascular elements within scaffolds, and in the shortest period of time, when compared to the other tested cell combinations. These differences were manifested by higher network complexity, more tube alignment and higher α-smooth muscle actin expression. Moreover, these generated microvessels further matured and developed during the 14-day incubation period within the three-dimensional microenvironment.ConclusionsThese data demonstrate optimal vascular network formation upon co-culture of microvascular endothelial cells and adipose-derived MSCs in vitro and constitute a significant step in appreciation of the potential of microvascular endothelial cells and MSCs in different tissue engineering applications that can also be advantageous in in vivo studies.

Blood stream infections (BSI) in severe burn patients—Early and late BSI: A 9-year study

Bloodstream infections (BSI) and sepsis are among the most common complications occurring in severe burn patients. This study was designed to evaluate changes in BSI pathogens over almost a decade in severe burn patients at Rambam Healthcare Campus, and BSI occurrence during early and late hospitalization periods. Retrospective computerized data was retrieved from all severe burn patients hospitalized in our institution during the years 2001-2009. BSI in the first week was defined as early BSI, and in the second week and beyond, late BSI. Of 159 severe burns patients, 74 had at least one BSI episode. Most first BSI episodes were diagnosed during the first week of hospitalization. In late BSI, an increased prevalence of resistant bacteria (methicillin-resistant Staphylococcus aureus [MRSA], carbapenem-resistant Klebsiella pneumoniae [CRKP], imipenem-resistant Pseudomonas aeruginosa [PSE-IMP]) and Candida spp. were observed. However, over the 9-year study period, only CRKP increased significantly. In summary, except for the sudden appearance and increase in CRKP (8% increase; p=0.045), we did not observe a significant change in the BSI pathogen profile over the 9-year period. Nevertheless, over the hospitalization period, there is a clear change in the BSI bacteria profile, especially after 4 weeks of hospitalization.

Novel biodegradable composite wound dressings with controlled release of antibiotics: Results in a guinea pig burn model

Approximately 70% of all people with severe burns die from related infections despite advances in treatment regimens and the best efforts of nurses and doctors. Silver ion-eluting wound dressings are available for overcoming this problem. However, there are reports of deleterious effects of such dressings due to cellular toxicity that delays the healing process, and the dressing changes needed 1-2 times a day are uncomfortable for the patient and time consuming for the stuff. An alternative concept in wound dressing design that combines the advantages of occlusive dressings with biodegradability and intrinsic topical antibiotic treatment is described herewith. The new composite structure presented in this article is based on a polyglyconate mesh and a porous poly-(dl-lactic-co-glycolic acid) matrix loaded with gentamicin developed to provide controlled release of antibiotics for three weeks. In vivo evaluation of the dressing material in contaminated deep second degree burn wounds in guinea pigs (n=20) demonstrated its ability to accelerate epithelialization by 40% compared to an unloaded format of the material and a conventional dressing material. Wound contraction was reduced significantly, and a better quality scar tissue was formed. The current dressing material exhibits promising results, does not require frequent bandage changes, and offers a potentially valuable and economic approach to treating the life-threatening complication of burn-related infections.

Multimodality gynecomastia repair by cross-chest power-assisted superficial liposuction combined with endoscopic-assisted pull-through excision.

Numerous methods of gynecomastia repair have been described to accomplish removal of breast tissue. Our multimodality surgical approach for the treatment of gynecomastia combines the use of power-assisted superficial cross-chest liposuction with direct pull-through excision of the breast parenchyma under endoscopic supervision. Seventeen patients, aging 17–39, underwent this multimodality approach. According to Simons grading, 3 patients had grade 1, 5 had grade 2a, 6 had grade 2b, and 3 had grade 3 gynecomastia. Power-assisted liposuction was performed with a 3- or 4-mm triple-hole cannula inserted through the contralateral periareolar medial incision to suction the contralateral prepectoral fatty breast. At the end of the liposuction, the fibrous tissue was easily pulled through the ipsilateral stab wound and excised under endoscopic control. Follow-up time ranged from 6 to 34 months. The amount of fat removed by liposuction varied from 100–800 mL per breast, and the amount of breast parenchyma removed by excision varied from 20–110 g. All patients recovered remarkably well. No complications were recorded. All patients were satisfied with their results. This technique enables an effective treatment of both the fatty and fibrous tissue of the male breast and avoids skin redundancy due to skin contraction. A smooth masculine breast contour is consistently achieved without the stigma of this type of surgery.

Hair removal using intense pulsed light (EpiLight): patient satisfaction, our experience, and literature review.

Applications for intense pulsed light (IPL) for hair removal are gaining favor among other methods, including lasers, because of its noninvasive nature, versatility regarding different skin and hair types, safety, and ease of use. Hair removal using IPL was performed from January 2002 to December 2003 on 108 consecutive patients. Eighty of these patients answered a questionnaire and were enrolled in the study group. The investigated parameters were hair and skin type, number of pulses, fluence, pulse duration, pulse delay, the filters used, and the treated area. The patient assessment of improvement (satisfaction) rate was graded from 1 to 5 points: 1, worse; 2, no improvement; 3, mild improvement; 4, good result; and 5, excellent result. The patients had between 1 and 13 treatments most of them during 2 to 6 sessions. Sixty-seven percent of the patients reported no complications. Prolonged erythema for more than 7 days was reported by 16.25%, blisters by 6.25%, temporary hyperpigmentation by 8.75%, leukotrichia was present in 1 case, and 1 case of persistent hypopigmentation was noted in a young girl. An increased number of complications and a decreased satisfaction rate were noted with higher skin types, but it was not statistically significant. Patients who underwent fewer treatments (1–3 treatments) were more satisfied compared with those who had more than 7 treatments (P < 0.02). Sixty percent of the patients rated their satisfaction to be good to excellent and 65% would ill recommend this treatment to their friends. To minimize the complication rate the authors found that the preset parameters should be adjusted at every treatment session according to the skin response at the previous one. Permanent hair removal cannot be guaranteed and it is not possible to predict the improvement rate. Nevertheless, based on patient satisfaction rate in this study, the authors recommend using IPL for hair removal.

Salvage of compromised free flaps in trauma cases with combined modalities

Using the microsurgical technique for reconstruction in trauma cases represents a challenge for the reconstructive surgeon. Several methods of salvaging of a compromised free flap have been reported, among them: intravenous heparin washing, thrombolitic therapy, thrombectomy, use of grafts and others. Here, we present our experience from nine cases and a review of the literature regarding the use of various modalities for free flap salvage in trauma cases, and their results. Data was collected from trauma cases in our institutions over a period of 2 years, where reconstruction was performed using microsurgical techniques, and where subsequent complications required some type of salvage procedure. The techniques that were used for the salvage included: intravascular irrigation with heparin, papaverine and lidocaine; administration of continuous intravenous heparin, use of the Fogarty catheter, flap washing with streptokinase, and adventitia stripping. The free flaps used were latissimus dorsi, serratus anterior, and the anteromedial thigh flap. Either vein or artery thromboses were identified during the procedure or immediately after surgery in seven patients. Two patients had prolonged spasms of the recipient artery with low flow. In all cases, the No. 2 Fogarty catheter was used for thrombectomy and also for release of the vessel spasm. There was only one complete failure among these patients, and partial necrosis was encountered in three. From our experience and review of the literature, we offer an algorithm for determining treatment strategies in a range of flap salvage situations.