The Urogenital Epithelium and Corporal Tissues Are the Primary Targets of Rejection in Penile Vascularized Composite Allotransplantation: A New Real-Time Tissue-Based Monitoring System.

BACKGROUND: Although significant surgical advances have been made in the form of microvascular surgery and autologous free tissue transfer, penile reconstruction still poses several difficult challenges. Although interest in penile vascularized composite allotransplantation has grown since the first attempted transplant in 2006, little is known regarding the kinetics of rejection and subsequent function of penile allografts. The penis contains multiple tissue types that are not qualified by the Banff 2007 vascularized composite allotransplantation classification system, including urogenital mucosal epithelium and erectile tissues. In this study, the authors investigate the propagation of rejection and the resultant function following rejection in rat and human penile tissues. METHODS: Rejected human and rat penile tissues were examined using an ex vivo real-time tissue-based derivative of the classic mixed lymphocyte reaction assay to determine the interactions occurring between en bloc penile tissues and peripheral blood mononuclear cells (autologous and allogeneic). Correlative in vivo heterotopic rat penile vascularized composite allotransplantation was used to correlate ex vivo findings. RESULTS: In both human and rat ex vivo systems and in vivo rat vascularized composite allotransplantation, the urethral mucosa was the first to undergo rejection-associated apoptosis. The urethral mucosa was the most immunogenic and led to the highest level of peripheral blood mononuclear cell proliferative generations in all systems, whereas the neural tissues of the penis remained immune privileged. CONCLUSION: These findings are the first to describe the kinetics of rejection in both human and rat penile vascularized composite allotransplantation and that the urethral mucosa is the most antigenic, suffering the highest level of rejection-associated apoptosis and peripheral blood mononuclear cell proliferative aggregation.

Muscle-Derived Stem Cell-Enriched Scaffolds Are Capable of Enhanced Healing of a Murine Volumetric Muscle Loss Defect.

BACKGROUND: Volumetric muscle loss secondary to traumatic or surgical causes can lead to functional and aesthetic impairments. The authors hypothesize that an implantable muscle-derived stem cell-enriched collagen scaffold could significantly augment muscle regeneration in a murine model of volumetric muscle loss. METHODS: Murine muscle-derived stem cells were isolated using a modified preplating technique and seeded onto type 1 collagen scaffolds to create the muscle-derived stem cell-enriched collagen scaffolds. Murine rectus femoris defects of 5 mm were created and randomized to one of three conditions (n = 6 per group): untreated controls, collagen scaffold only, and muscle-derived stem cell-enriched collagen scaffolds. In vivo muscle healing was quantified using micro-computed tomography. Muscle explants were analyzed using standard histology and whole-mount immunofluorescence at 8 weeks. RESULTS: In vivo experiments demonstrated significantly greater quadriceps cross-sectional area in the muscle-derived stem cell-enriched collagen scaffold group compared with controls on micro-computed tomography (0.74 ± 0.21 versus 0.55 ± 0.06 versus 0.49 ± 0.04 ratio of experimental to naive quadriceps cross-sectional area; p < 0.05). Muscle explants of the muscle-derived stem cell-enriched collagen scaffold group demonstrated significantly higher cellular density compared with controls (1185 ± 360 versus 359 ± 62 versus 197 ± 68 nuclei/high-power field; p < 0.01). Immunofluorescence for laminin and myosin heavy chain confirmed formation of organized muscle fibers within the defect of the muscle-derived stem cell-enriched collagen scaffold group only. However, appreciable confocal colocalization of myosin heavy chain with green fluorescent protein expression was low. CONCLUSIONS: The results of this study indicate that muscle-derived stem cell-enriched scaffolds significantly improved skeletal muscle regeneration in a murine muscle defect model. Based on the low fluorescent colocalization, host progenitor cells appear to contribute significantly to intradefect myogenesis, suggesting that deployment of a viable muscle-derived stem cell-enriched scaffold stimulates a regenerative mitogen response in native tissues.

Desensitization and Prevention of Antibody-Mediated Rejection in Vascularized Composite Allotransplantation by Syngeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Candidates for vascularized composite allotransplantation (VCA) are frequently sensitized, putting them at risk for antibody-mediated rejection. Current desensitization strategies are imperfect and require a living-donor setting. Here we investigated the impact of sensitization on and the efficacy of a desensitization protocol utilizing syngeneic hematopoietic stem cell transplantation (HSCT) to prevent antibody-mediated rejection in VCA. METHODS: Skin transplants from Dark Agouti to Lewis rats were performed for sensitization. Orthotopic hind limb transplants from Dark Agouti donors were performed to sensitized and nonsensitized recipients, and the animals were treated with either daily tacrolimus or no immunosuppression. A desensitization protocol consisting of total body irradiation, fludarabine, and syngeneic HSCT was applied to sensitized animals. Graft rejection was monitored by clinical assessment and histological analysis. Serum levels of donor-specific antibodies (DSA IgG) were measured using flow cytometry. RESULTS: Sensitized recipients exhibited accelerated rejection by 5.5 ± 1.2 days without immunosuppression and 10.2 ± 3.6 days with daily tacrolimus compared with 8.7 ± 1.2 days and longer than 30 days in nonsensitized recipients, respectively. Serum levels of DSA IgG were markedly elevated (37.3 ± 3.34-fold from baseline) in sensitized recipients after VCA and correlated with histologic evidence of rejection and C4d deposition. Desensitization significantly reduced DSA compared with sensitized controls (2.6 ± 0.5-fold vs 6.0 ± 1.2-fold, P < 0.01) and along with daily tacrolimus led to improved VCA survival longer than 30 days without evidence of C4d deposition (n = 6). CONCLUSIONS: In summary, sensitization leads to accelerated rejection of VCA, and syngeneic HSCT combined with conventional immunosuppression effectively reduces DSA and improves allograft survival in sensitized rats.

Vascular Endothelial Growth Factor Induction of Muscle-Derived Stem Cells Enhances Vascular Phenotype While Preserving Myogenic Potential.

BACKGROUND: Previous work by our group and other laboratories have revealed that muscle-derived stem cells (MDSCs) may contain both myogenic and endothelial progenitors, making MDSCs a promising option for skeletal muscle regeneration. The purpose of this study was to investigate the impact of vascular endothelial growth factor (VEGF) induction on the vascular and myogenic potential of MDSCs. METHODS: Muscle-derived stem cells were isolated from 4- to 8-week-old C57BL/6J mice using a preplate technique and recombinant human VEGFa was used as the induction agent. Cellular proliferation and migration were assessed using serial imaging and wound healing assays, respectively. Myosin heavy chain staining was performed to assess MDSC myotube formation. Vascular potential of MDSCs was measured by expression of CD31 and in vitro capillary tube formation. RESULTS: Vascular endothelial growth factor stimulation led to a dose-dependent increase in MDSC proliferation (P < 0.05) and migration kinetics (P < 0.01). Control MDSCs had low levels of baseline expression of CD31, which was significantly upregulated by VEGF stimulation. Similarly, MDSCs demonstrated a basal capability for capillary tube formation, which was significantly increased after VEGF induction as evidenced by increased branches (5.91 ± 0.58 vs 9.23 ± 0.67, P < 0.01) and total tube length (11.73 ± 0.97 vs 18.62 ± 1.57 mm, P < 0.01). Additionally, the myogenic potential of MDSCs as measured by fusion index remained unchanged with increasing concentration of VEGF up to 250 ng/mL (P = 0.77). CONCLUSIONS: Vascular endothelial growth factor induction enhances MDSC proliferation, migration, and endothelial phenotypes without negatively impacting myogenic potential. These results suggest that VEGF stimulation may improve vascularization of MDSC-based strategies for skeletal muscle regeneration.

Penile transplantation: an emerging option for genitourinary reconstruction.

Penile transplantation is an emerging option for patients with severe genital defects not amenable to traditional reconstructive options. In this article, we discuss the burgeoning problem of severe male genitourinary trauma in the military, the limitations of traditional reconstructive options in addressing these problems, and the potential for penile transplantation to provide improved outcomes. We also review the preclinical research and limited worldwide experience with penile transplantation to date, including lessons learned, and discuss the many important technical, logistical, and ethical considerations pertaining to penile transplantation that must be addressed to maximize the likelihood of successful implementation.

Ex Vivo Model of Human Penile Transplantation and Rejection: Implications for Erectile Tissue Physiology.

BACKGROUND: Penile transplantation is a potential treatment option for severe penile tissue loss. Models of human penile rejection are lacking. OBJECTIVE: Evaluate effects of rejection and immunosuppression on cavernous tissue using a novel ex vivo mixed lymphocyte reaction (MLR) model. DESIGN, SETTING, AND PARTICIPANTS: Cavernous tissue and peripheral blood mononuclear cells (PBMCs) from 10 patients undergoing penile prosthesis operations and PBMCs from a healthy volunteer were obtained. Ex vivo MLRs were prepared by culturing cavernous tissue for 48h in media alone, in media with autologous PBMCs, or in media with allogenic PBMCs to simulate control, autotransplant, and allogenic transplant conditions with or without 1µM cyclosporine A (CsA) or 20nM tacrolimus (FK506) treatment. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Rejection was characterized by PBMC flow cytometry and gene expression transplant array. Cavernous tissues were evaluated by histomorphology and myography to assess contraction and relaxation. Data were analyzed using two-way analysis of variance and unpaired Student t test. RESULTS AND LIMITATIONS: Flow cytometry and tissue array demonstrated allogenic PBMC activation consistent with rejection. Rejection impaired cavernous tissue physiology and was associated with cellular infiltration and apoptosis. CsA prevented rejection but did not improve tissue relaxation. CsA treatment impaired relaxation in tissues cultured without PBMCs compared with media and FK506. Study limitations included the use of penile tissue with erectile dysfunction and lack of cross-matching data. CONCLUSIONS: This model could be used to investigate the effects of penile rejection and immunosuppression. Additional studies are needed to optimize immunosuppression to prevent rejection and maximize corporal tissue physiology. PATIENT SUMMARY: This report describes a novel ex vivo model of human penile transplantation rejection. Tissue rejection impaired erectile tissue physiology. This report suggests that cyclosporin A might hinder corporal physiology and that other immunosuppressant agents, such as FK506, might be better suited to penile transplantation.

Regulation of ADSC Osteoinductive Potential Using Notch Pathway Inhibition and Gene Rescue: A Potential On/Off Switch for Clinical Applications in Bone Formation and Reconstructive Efforts.

BACKGROUND: Although there has been tremendous research in the ability of mesenchymal-derived adipose derived stem cells (ADSCs) to form bone, less is known regarding the molecular mechanisms that regulate the osteogenic potential of ADSCs. Notch, which consists of a key family of regulatory ligands involved in bone formation, is expressed in the bone marrow-derived mesenchymal stem cell niche and is critical for proliferation, migration, and ultimately osseous differentiation. The authors investigate how Notch impacts ADSC proliferation and osteogenic differentiation to determine a translatable application of these cells in bone regeneration. METHODS: Enriched ADSC populations were isolated from tissue and examined for their ability to respond to Notch pathway signaling events. Proliferation, viability, extracellular matrix deposition, and osteoinduction were assessed following Notch activation and inhibition. Notch pathway rescue was conducted using a lentiviral vector encoding a downstream Notch-1 intracellular domain (NICD). RESULTS: Proliferation, osteogenic induction, and the ability to form bone elements were reduced following Notch inhibition (p < 0.05). However, ADSCs, while in the presence of the Notch inhibition, were able to be rescued following lentiviral transduction with NICD, restoring osteogenic potential at both the molecular and cellular functional levels (p < 0.05). CONCLUSIONS: These data suggest a potential translatable "on/off switch," using endogenous Notch signaling to regulate the proliferation, differentiation, and osteogenic potential of ADSCs. Although Notch inhibition reduced ADSC proliferation and down-regulated osteoinduction, targeted gene therapy and the delivery of the downstream NICD peptide restored bone formation, suggesting pragmatic clinical utility of ADSCs for bone regeneration.

Does negative pressure wound therapy applied to closed incisions following ventral hernia repair prevent wound complications and hernia recurrence? A systematic review and meta-analysis.

BACKGROUND: Despite advances in surgical technique, ventral hernia repair (VHR) remains associated with significant postoperative wound complications. OBJECTIVE: A systematic review and meta-analysis was performed to identify whether the application of negative pressure wound therapy to closed incisions (iNPWT) following VHR reduces the risk of postoperative wound complications and hernia recurrence. METHODS: The PubMed/MEDLINE, EMBASE and SCOPUS databases were searched for studies published through October 2015. Publications that met the following criteria were included: adult patients undergoing VHR; comparison of iNPWT with conventional dressings; and documentation of wound complications and/or hernia recurrence. The methodological quality of included studies was independently assessed using the Methodological Index for Non-Randomized Studies guidelines. Outcomes assessed included surgical site infection (SSI), wound dehiscence, seroma, and hernia recurrence. Meta-analysis was performed to obtain pooled ORs. RESULTS: Five retrospective cohort studies including 477 patients undergoing VHR were included in the final analysis. The use of iNPWT decreased SSI (OR 0.33 [95% CI 0.20 to 0.55]; P<0.0001), wound dehiscence (OR 0.21 [95% CI 0.08 to 0.55]; P=0.001) and ventral hernia recurrence (OR 0.24 [95% CI 0.08 to 0.75]; P=0.01). There was no statistically significant difference in the incidence of seroma formation (OR 0.59 [95% CI 0.27 to 1.27]; P=0.18). CONCLUSION: For patients undergoing VHR, current evidence suggests a decreased incidence in wound complications using incisional NPWT compared with conventional dressings.

HISTORIQUE: Malgré les progrès des techniques chirurgicales, la réparation de la hernie ventrale (RHV) s'associe encore à des complications importantes de la plaie postopératoire. OBJECTIF: Les chercheurs ont réalisé une analyse systématique et une méta-analyse pour déterminer si la thérapie par pression négative sur des incisions fermées (TPNiF) après la RHV réduit le risque de complications postopératoires des plaies et la récurrence des hernies. MÉTHODOLOGIE: Les chercheurs ont exploré les bases de données PubMed/MEDLINE, EMBASE et SCOPUS pour trouver des études publiées jusqu'en octobre 2015. Ils ont retenu les publications qui respectaient les critères suivants : patients adultes ayant subi une RHV, comparaison de la TPNiF avec des pansements classiques et les rapports sur les complications des plaies ou la récurrence des hernies. Ils ont évalué de manière indépendante la qualité méthodologique des études retenues à l'aide des directives de l'indice méthodologique des études non aléatoires. Ils ont évalué les résultats suivants : l'infection au foyer de l'opération (IFO), la déhiscence de la plaie, le sérome et la récurrence des hernies. Ils ont effectué une méta-analyse pour obtenir les rapports de cote (RC) regroupés. RÉSULTATS: Les chercheurs ont retenu cinq études de cohorte rétrospectives, y compris 477 patients qui avaient subi une RHV, dans l'analyse définitive. Le recours à la TPNiF réduisait l'IFO (RC 0,33 [95 % IC 0,20 à 0,55]; P<0,0001), la déhiscence de la plaie (RC 0,21 [95 % IC 0,08 à 0,55]; P=0,001) et la récurrence de la hernie ventrale (RC 0,24 [95 % IC 0,08 à 0,75]; P=0,01). Ils n'ont pas constaté de différence statistiquement significative dans l'incidence de formation de séromes (RC 0,59 [95 % IC 0,27 à 1,27]; P=0,18). CONCLUSION: Pour les patients qui subissent une RHV, les données actuelles indiquent que l'incidence des complications des plaies est moins élevée si on utilise la TPNiF plutôt que les pansements classiques.

Transplantation of an LGR6+ Epithelial Stem Cell-Enriched Scaffold for Repair of Full-Thickness Soft-Tissue Defects: The In Vitro Development of Polarized Hair-Bearing Skin.

BACKGROUND: Recent literature has shown that full-thickness wounds, devoid of the stem cell niche, can subsequently be reconstructed with functional skin elements following migration of the LGR6 epithelial stem cell into the wound bed. In this study, the authors use a variety of LGR6 epithelial stem cell-seeded scaffolds to determine therapeutic utility and regenerative potential in the immediate reconstruction of full-thickness wounds. METHODS: Isolated LGR6 epithelial stem cells were seeded onto a spectrum of acellular matrices and monitored in both in vitro and in vivo settings to determine their relative capacity to regenerate tissues and heal wounds. RESULTS: Wound beds containing LGR6 stem cell-seeded scaffolds showed significantly augmented rates of healing, epithelialization, and hair growth compared with controls. Gene and proteomic expression studies indicate that LGR6 stem cell-seeded constructs up-regulate WNT, epidermal growth factor, and angiogenesis pathways. Finally, the addition of stromal vascular fraction to LGR6 stem cell-seeded constructs induces polarized tissue formation, nascent hair growth, and angiogenesis within wounds. CONCLUSIONS: LGR6 stem cells are able to undergo proliferation, differentiation, and migration following seeding onto a variety of collagen-based scaffolding. In addition, deployment of these constructs induces epithelialization, hair growth, and angiogenesis within wound beds. The addition of stromal vascular fraction to LGR6 stem cell-containing scaffolds initiated an early form of tissue polarization, providing for the first time a clinically applicable stem cell-based construct that is capable of the repair of full-thickness wounds and hair regeneration. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Intraoperative Use of Vasopressors Is Safe in Head and Neck Free Tissue Transfer.

BACKGROUND: The purpose of this study is to identify whether intraoperative use of vasoactive medications increases the risk of free flap failure or complications through a systematic review and meta-analysis. MATERIALS AND METHODS: PubMed/MEDLINE, EMBASE, and Scopus databases were searched for studies published through January 2015. English publications that met the following criteria were included: (1) adult patients undergoing head and neck free flap reconstruction; (2) comparison of patients with and without intraoperative vasopressor administration; and (3) documentation of flap failure rate and/or flap complications. The primary outcome was the incidence of flap failure. The secondary outcome was the incidence of overall flap complications. Meta-analysis was performed to obtain pooled odds ratios (ORs) of the effect of intraoperative use of vasopressors on flap failure and complication rates. RESULTS: Four cohort studies met inclusion criteria. All studies were of high methodological quality with an average Methodological Index for Non-Randomized Studies score of 18.75 (range 16-23). A total of 933 patients undergoing head and neck free flap reconstruction were included. Meta-analysis demonstrated no statistically significant difference in the incidence of flap failure (2.9 vs. 3.6%; OR, 0.68; 95% confidence interval [CI], 0.23-1.99; p = 0.48) or incidence of flap complications (16.8 vs. 18.6%; OR, 0.92; 95% CI, 0.60-1.42; p = 0.71). CONCLUSION: Based on the current evidence, intraoperative use of vasopressors has no impact on the incidence of flap failure or flap complications.

What Effect Does Self-Citation Have on Bibliometric Measures in Academic Plastic Surgery?

BACKGROUND: Research productivity plays a significant role in academic promotions. Currently, various bibliometric measures utilizing citation counts are used to judge an author's work. With increasing numbers of journals, numbers of open access publications, ease of online submission, and expedited indexing of accepted manuscripts, it is plausible that an author could influence his/her own bibliometric measures through self-citation. The purpose of this study was to determine the impact of self-citation in academic plastic surgery. METHODS: A cohort of full-time academic plastic surgeons was identified from 9 U.S. plastic surgery training programs. For all included faculty, academic rank was retrieved from department/division websites, and bibliometric measures were assessed using a subscription bibliographic citation database (Scopus, Reed Elsevier, London, UK). Bibliometric measures included the Hirsch index (h-index, the number of publications h which are cited ≥ h times), total number of publications, and total number of citations. The h-index and total number of citations were collected with and without self-citations. Percent changes in the h-index and total citations were calculated after removal of self-citations and compared across academic ranks and levels of research productivity (total publications, h-index, and total citations). RESULTS: The study cohort consisted of 169 full-time academic plastic surgeons. The h-index and total citations experienced decreases of 2.8 ± 5.0% (P < 0.0001) and 4.5 ± 4.6% (P < 0.0001), respectively, after correction for self-citation. More than half of the cohort (n = 113, 67%) did not experience a change in the h-index after removal of self-citations. These decreases did not vary across academic rank. Surgeons who self-cited at rates greater than 5% were 9.8 times more likely (95% confidence interval, 4.5-21.9; P < 0.001) to have their h-index change as a result of self-citation (after adjusting for academic rank). There were weak correlations between percent decreases in the h-index and total citations and various biblimoteric measures (total publications, h-index, total citations; r < 0.32). CONCLUSIONS: Self-citation has a minor impact on common bibliometric measures in academic plastic surgery. The influence of self-citation is consistent across academic ranks and increasing levels of bibliometric measures, suggesting that authors are not manipulating the system with increasing experience.

Transplantation of the LGR6+ epithelial stem cell into full-thickness cutaneous wounds results in enhanced healing, nascent hair follicle development, and augmentation of angiogenic analytes.

BACKGROUND: The recently discovered leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6+) epithelial stem cell located within the follicular bulge of the adnexal compartment is capable of producing all cellular lineages of the skin. In this study, the authors sought to determine whether these cells can be transplanted for use as a type of cellular therapy for the repair of full-thickness wounds in which the native stem cell niche has been obliterated. METHODS: Full-thickness murine skin was harvested and LGR6(+GFP) epithelial stem cells were isolated using fluorescence-activated cell sorting. This enriched epithelial stem cell population was then transplanted by means of local injection into wound beds on the dorsum of nude mice. Viability, migration, healing, the development of nascent hair follicles, and gene and proteomic expression studies were performed to determine whether the engraftment of LGR6(+GFP) epithelial stem cells enhanced healing when compared with controls. RESULTS: Wound beds receiving LGR6(+GFP) epithelial stem cells showed enhanced healing; nascent follicle growth; and augmentation of the Wnt, vascular endothelial growth factor, epidermal growth factor, and platelet-derived growth factor pathways when compared with controls. CONCLUSIONS: The LGR6+ epithelial stem cells appear to hold great promise for the development of a clinically useful stem cell­based therapy for the repair of full-thickness wounds and hair regeneration. These results indicate that transplantation of LGR6+ epithelial stem cells promotes epithelialization, hair growth, and angiogenesis in tissues destined for scar formation.

Human gut microbiome adopts an alternative state following small bowel transplantation.

Small bowel transplants provide an exceptional opportunity for long-term study of the microbial ecology of the human small bowel. The ileostomy created at time of transplant for ongoing monitoring of the allograft provides access to samples of ileal effluent and mucosal biopsies. In this study, we used qPCR to assay the bacterial population of the small bowel lumen of 17 small bowel transplant patients over time. Surprisingly, the posttransplant microbial community was found to be dominated by Lactobacilli and Enterobacteria, both typically facultative anaerobes. This represents an inversion of the normal community that is dominated instead by the strictly anaerobic Bacteroides and Clostridia. We found this inverted community also in patients with ileostomies who did not receive a transplant, suggesting that the ileostomy itself is the primary ecological determinant shaping the microbiota. After surgical closure of the ileostomy, the community reverted to the normal structure. Therefore, we hypothesized that the ileostomy allows oxygen into the otherwise anaerobic distal ileum, thus driving the transition from one microbial community structure to another. Supporting this hypothesis, metabolomic profiling of both communities demonstrated an enrichment for metabolites associated with aerobic respiration in samples from patients with open ileostomies. Viewed from an ecological perspective, the two communities constitute alternative stable states of the human ileum. That the small bowel appears to function normally despite these dramatic shifts suggests that its ecological resilience is greater than previously realized.