Radioterapia en colgajos peroneos. Revisión de la densidad ósea y complicaciones en colgajos peroneos irradiados frente a no irradiados / Effects of radiotherapy in fibular free flaps. Study of bone density and complications in irradiated fibular flaps vs no irradiated

INTRODUCCIÓN: El colgajo peroneo es actualmente una de las técnicas más elegidas para la reconstrucción de defectos mandibulares. Muchos de los pacientes reconstruidos con este colgajo presentan patología oncológica que precisa tratamiento adyuvante con radioterapia. Los efectos adversos de la radioterapia sobre los tejidos son conocidos, pudiendo desembocar en osteoradionecrosis. El objetivo de este estudio es el análisis de la densidad ósea de los colgajos peroneos en los pacientes que han recibido radioterapia frente a los que no y su posible correlación con las complicaciones posteriores. MATERIAL Y MÉTODOS: Realizamos un estudio retrospectivo de los colgajos peroneos realizados por nuestro servicio desde enero 2011 hasta diciembre 2016. Los criterios de inclusión de los pacientes fue el haber sido reconstruidos con un colgajo peroneo y disponer de un TC de control a los tres meses postradioterapia en caso de haberla precisado. Se recogió la edad, densidad ósea peronea, desviación estándar cuerpo mandibular remanente y colgajo peroneo, así como las complicaciones postoperatorias. Los datos extraídos fueron analizados a través del Software SAS (Statistical Analisys System). RESULTADOS: En total se recogieron 61 pacientes, siendo la media de edad de 54,46 años. De estos 61 pacientes se excluyeron 11 al no presentar TC de control; de los 50 pacientes restantes, 27 recibieron tratamiento radioterápico adyuvante (54 %), mientras que 23 no la recibieron (46 %). En los pacientes irradiados, un alto porcentaje (84,62 %) recibieron altas dosis de radioterapia (> 60 Gy). Se encontraron diferencias estadísticamente significativas (p < 0,05) respecto a la edad y la desviación estándar de la densidad ósea del cuerpo mandibular remanente, mientras que en el resto de los parámetros no se encontraron diferencias estadísticamente significativas. Los pacientes irradiados presentaron un mayor porcentaje de complicaciones frente a los pacientes no irradiados, sin diferencias estadísticamente significativas. Discusión: Las nuevas técnicas de radioterapia de intensidad modulada permiten una dosis relativamente uniforme en un objetivo, evitando altas dosis en los tejidos circuncidantes. A pesar del avance en las técnicas de radioterapia, actualmente en la mayoría de centros se continúa realizando un tratamiento completo del volumen del lecho tumoral, que incluye en el campo de irradiación el colgajo con el que se reconstruye. Los casos de osteonecrosis mandibular que se presentaron fueron en pacientes irradiados y a nivel del cuerpo mandibular remanente. La densidad ósea a nivel del colgajo peroneo sí que presentó diferencias estadísticamente significativas en pacientes irradiados frente a no irradiados; este aspecto puede influir en el índice de complicaciones. CONCLUSIONES: Consideramos, con los resultados del estudio, plantear nuevos estudios prospectivos para valorar la necesidad de irradiar el tejido óseo del colgajo peroneo como parte del volumen del lecho tumoral, siendo que este tejido no ha estado en contacto con el tumor primario, ante el mayor índice de complicaciones asociados a la radioterapia en estos tejidos

INTRODUCTION: The fibular flap is currently one of the most chosen techniques for the reconstruction of mandibular defects. Many of the patients reconstructed with this flap present oncological pathology that requires adjuvant treatment with radiotherapy. The adverse effects of radiotherapy on tissues are known, and can lead to osteoradionecrosis. The aim of this study is to analyze the bone density of peroneal flaps in patients who have received radiotherapy against those who do not and their possible correlation with subsequent complications. MATERIAL AND METHODS: We designed a retrospective study of the fibular flaps performed by our service from January 2011 to December 2016. The criteria for inclusion of patients was to have been reconstructed with a peroneal flap and have a control CT at 3 months after radiotherapy. Age, bone density, standard deviation and further complications were extracted. The extracted data was analyzed through the SAS (statistical Analisys System) Software. RESULTS: 61 patients were collected; the mean age was of 54.46 years. Of these 61 patients were excluded 11 by not presenting control TC, of the 50 patients remaining, 27 received adjuvant radiotherapy treatment (54 %) while 23 did not receive (46 %). In irradiated patients a high percentage (84.62 %) received high doses of radiotherapy (> 60 Gy). Statistically significant differences were found (P < 0.05) with respect to age and standard deviation of bone density of the remaining mandibular body, while in the rest of the parameters no statistical significant differences were found. Irradiated patients presented a higher percentage of complications compared to non-irradiated patients without statistically significant differences. Discussion: The new techniques of intensity-modulated radiotherapy allow a relatively uniform dose in a target, avoiding high doses in the circumcising tissues. Despite the progress in radiotherapy techniques, currently in most centers, it continues to perform a complete treatment of the volume of the tumor bed that includes in the field of irradiation the flap with which it is reconstructed. The cases of mandibular osteonecrosis that were presented were in irradiated patients and in the remaining mandible. Bone density at the level of the fibular flap showed statistically significant differences in irradiated patients compared to non-irradiated, this aspect may influence in the index of complications. CONCLUSIONS: We consider to propose new prospective studies to assess the need to irradiate the bone tissue of the fibular flap as part of the volume of the tumor bed, knowing that this tissue has not been in contact with the primary tumor, and the high index of complications associated with radiotherapy

Immunological and inflammatory mapping of vascularized composite allograft rejection processes in a rat model.

BACKGROUND: Hand and face vascularized composite allotransplantation (VCA) is an evolving and challenging field with great opportunities. During VCA, massive surgical damage is inflicted on both donor and recipient tissues, which may contribute to the high VCA rejection rates. To segregate between the damage-induced and rejection phase of post-VCA responses, we compared responses occurring up to 5 days following syngeneic versus allogeneic vascularized groin flap transplantations, culminating in transplant acceptance or rejection, respectively. METHODS: The immune response elicited upon transplantation of a syngeneic versus allogeneic vascularized groin flap was compared at Post-operative days 2 or 5 by histology, immunohistochemistry and by broad-scope gene and protein analyses using quantitative real-time PCR and Multiplex respectively. RESULTS: Immune cell infiltration began at the donor-recipient interface and paralleled expression of a large group of wound healing-associated genes in both allografts and syngrafts. By day 5 post-transplantation, cell infiltration spread over the entire allograft but remained confined to the wound site in the syngraft. This shift correlated with upregulation of IL-18, INFg, CXCL9, 10 and 11, CCL2, CCL5, CX3CL1 and IL-10 in the allograft only, suggesting their role in the induction of the anti-alloantigen adaptive immune response. CONCLUSIONS: High resemblance between the cues governing VCA and solid organ rejection was observed. Despite this high resemblance we describe also, for the first time, a damage induced inflammatory component in VCA rejection as immune cell infiltration into the graft initiated at the surgical damage site spreading to the entire allograft only at late stage rejection. We speculate that the highly inflammatory setting created by the unique surgical damage during VCA may enhance acute allograft rejection.

Adipose-Derived Stem Cells Protect Skin Flaps against Ischemia/Reperfusion Injury via IL-6 Expression.

Flap necrosis is the most frequent postoperative complication encountered in reconstructive surgery. We elucidated whether adipose-derived stem cells (ADSCs) and their derivatives might induce neovascularization and protect skin flaps during ischemia/reperfusion (I/R) injury. Flaps were subjected to 3 hours of ischemia by ligating long thoracic vessels and then to blood reperfusion. Qtracker-labeled ADSCs, ADSCs in conditioned medium (ADSC-CM), or ADSC exosomes (ADSC-Exo) were injected into the flaps. These treatments led to significantly increased flap survival and capillary density compared with I/R on postoperative day 5. IL-6 levels in the cell lysates or in conditioned medium were significantly higher in ADSCs than in Hs68 fibroblasts. ADSC-CM and ADSC-Exo increased tube formation. This result was corroborated by a strong decrease in skin repair after adding IL-6-neutralizing antibodies or small interfering RNA for IL-6 ADSCs. ADSC transplantation also increased flap recovery in I/R injury of IL-6-knockout mice. IL-6 was secreted from ADSCs through signal transducer and activator of transcription phosphorylation, and then IL-6 stimulated angiogenesis and enhanced recovery after I/R injury by the classic signaling pathway. The mechanism of skin recovery includes the direct differentiation of ADSCs into endothelial cells and the indirect effect of IL-6 released from ADSCs. ADSC-CM and ADSC-Exo could be used as off-the-shelf products for this therapy.

Compartmentalization of immune responses during Staphylococcus aureus cranial bone flap infection.

Decompressive craniectomy is often required after head trauma, stroke, or cranial bleeding to control subsequent brain swelling and prevent death. The infection rate after cranial bone flap replacement ranges from 0.8% to 15%, with an alarming frequency caused by methicillin-resistant Staphylococcus aureus, which is problematic because of recalcitrance to antibiotic therapy. Herein we report the establishment of a novel mouse model of S. aureus cranial bone flap infection that mimics several aspects of human disease. Bacteria colonized bone flaps for up to 4 months after infection, as revealed by scanning electron microscopy and quantitative culture, demonstrating the chronicity of the model. Analysis of a human cranial bone flap with confirmed S. aureus infection by scanning electron microscopy revealed similar structural attributes as the mouse model, demonstrating that it closely parallels structural facets of human disease. Inflammatory indices were most pronounced within the subcutaneous galeal compartment compared with the underlying brain parenchyma. Specifically, neutrophil influx and chemokine expression (CXCL2 and CCL5) were markedly elevated in the galea, which demonstrated substantial edema on magnetic resonance images, whereas the underlying brain parenchyma exhibited minimal involvement. Evaluation of immune mechanisms required for bacterial containment and inflammation revealed critical roles for MyD88-dependent signaling and neutrophils. This novel mouse model of cranial bone flap infection can be used to identify key immunologic and therapeutic mechanisms relevant to persistent bone flap infection in humans.

Lentiviral transduction of face and limb flaps: implications for immunomodulation of vascularized composite allografts.

BACKGROUND: Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk-to-benefit ratio of vascularized composite allografts. Abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease alloreactivity and permit the induction of immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing transient gene expression. The authors hypothesized that transduction, integration, and long-term expression of transgenes in a vascularized composite allograft could be achieved using lentiviral vectors. METHODS: Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue flap's cellular architecture was performed using a luc-enhanced green fluorescent protein (eGFP) human immunodeficiency virus-1-based lentiviral vector. Ex vivo injections of rat superficial inferior epigastric artery flaps with the viral vector were performed intraarterially, intramuscularly, and intradermally. RESULTS: Quantifiable reporter expression by flow cytometry (fluorescence-activated cell sorting) analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibited broad tropism and allowed transgene expression in relevant cell lines and throughout the flaps. Ex vivo intradermal transfection resulted in genomic integration and long-term constitutive gene expression (>150 days). Similarly, efficient intradermal transfection of face and hand flaps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection. CONCLUSIONS: Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.

Short-term application of doxorubicin chemotherapy immunosuppressive side effects for composite tissue allotransplantation.

BACKGROUND: Adjuvant chemotherapy is often required for the treatment of bone cancers after tumor resection, which often results in a large continuity defect. The immunosuppressive side effects could instead be exploited to allow immediate reconstruction with a composite tissue allograft (CTA) that would provide for replacement of tissues. We used a short course of doxorubicin to achieve a novel method of immunosuppression in a rat model undergoing CTA to create an immunological environment for allograft survival. MATERIALS & METHODS: The Institutional Animal Care and Use Committee-approved protocol consisted of 3 experimental groups. Groups 2 and 3 consisted of Brown Norway rats (n = 5) as allograft donors and Lewis rats (n = 5) as transplant recipients. An abdominal wall CTA was harvested off the superficial inferior epigastric vessels. Doxorubicin therapy was administered in group 3 animals. Survival of the CTA was assessed by physical examination and histological analysis. RESULTS: Allotransplant without treatment showed complete clinical and histologic rejection by day 7. Allotransplant rats treated with doxorubicin had clinically and histologically normal grafts through day 10. Kaplan-Meier survival analysis showed a statistically significant difference, with increased CTA survival time to end point with doxorubicin treatment, from a mean of 8.8 days in group 2 to 16.4 days in group 3. CONCLUSIONS: Allotransplant flaps without treatment developed complete clinical and histological rejection. The allotransplant group which received doxorubicin showed a delay of allograft rejection with an 86% increased CTA graft survival time. This demonstrates the feasibility of the immunosuppression side effect caused by chemotherapy to prevent rejection of a CTA.

Vaginal reconstruction with two lower abdominal skin flaps in rabbits: histological and macroscopic evaluation.

OBJECTIVE: To evaluate the use of two skin flaps of the lower abdominal wall in the creation of a cylindrical conduit in vaginal reconstruction surgery in rabbits, through macroscopic and histological analysis. STUDY DESIGN: An experimental study was performed in 16 female New Zealand rabbits, consisting of the use of two rectangular-shaped skin flaps of the lower abdominal wall measuring 1cm longitudinally and 3 cm transversely anastomosed to each other through continuous suture of the edges of the two flaps to create a tube. Hysterectomy and excision of the vaginal vault were performed, and the skin tube was anastomosed to the remaining vaginal stump with separate points of polyglycolic acid 4.0. Animals were divided into 4 groups according to the euthanasia at 2, 4, 8 and 12 weeks, when after excision of the neovagina, macroscopic and histological evaluation with hematoxylin-eosin and Masson trichrome were performed. RESULTS: Of 16 operated rabbits, only 1 presented partial abdominal wall dehiscence, not compromising the flap viability. The macroscopic analysis of the vaginal conduit showed that it was kept open throughout the experimental steps, with a good patency and gauge, showing a slight retraction in the skin conduit length of no statistical significance. In the histopathological analysis, a local inflammatory process in the anastomosis was observed, which was larger in the early evaluation but decreased in late evaluations, as well as the local fibrosis process. Integration of the vaginal and skin epithelia was made with no alterations in their primary characteristics. CONCLUSION: The use of two skin flaps of the lower abdominal wall as a vaginal conduit presented good integration between skin and vaginal tissue with minimal length retraction, kept the patency during evaluations and did not show strictures, presenting good local healing and a low rate of complications.

Vascular and cellular events in post-mastectomy seroma: an immunohistochemical study.

This study aimed to describe the vascular and cellular histopathological changes that occurred in post-mastectomy seroma in an animal model. Unilateral mastectomies were conducted on 45 female albino rabbits. On day seven, the skin flap and the underlying tissues of the mastectomy regions were dissected and processed for histopathological examination using immunohistochemical staining of the T- and B-lymphocytes and macrophages (CD3, CD20, and CD68 respectively), and the vascular endothelia. The post-mastectomy regions in the seroma group showed a large number of inflammatory cells and newly formed blood vessels that lost the integrity of their endothelial cell linings, as revealed by the von Willebrand factor staining, as well the basement membrane, as revealed by the histochemical stain. The post-mastectomy seroma beds showed many CD3 and CD20+ve lymphocytes and CD68+ve macrophages. These macrophages were producing angiogenic factors, resulting in the persistent and continuous formation of new blood vessels. These new blood vessels were defective and represented an underlying cause of seroma formation.

Evidence that FoxP3+ regulatory T cells may play a role in promoting long-term acceptance of composite tissue allotransplants.

BACKGROUND: FoxP3/CD4/CD25 regulatory T cells (Treg) play an important role in maintaining peripheral tolerance and are potent suppressors of T-cell activation. In this study, we evaluated the role of Treg in peripheral tolerance to composite tissue allografts (CTA). METHODS: Mixed allogeneic chimeric rats were prepared by preconditioning recipients with anti-αß-T-cell receptor monoclonal antibody followed by total body irradiation. Animals received T-cell-depleted August Copenhagen Irish bone marrow cells followed by antilymphocyte serum and FK-506. A modified osteomyocutaneous hindlimb flap composed of bone and all limb tissue components was placed in animals with chimerism greater than or equal to 1% on day 28. Recipients with CTA surviving more than or equal to 6 months were evaluated for Treg. Skin samples from tolerant long-term allogeneic transplanted, syngeneic transplanted, rejected, and naïve animals were immunostained with fluorochrome-conjugated anti-FoxP3 and anti-CD4 monoclonal antibody and visualized under a laser confocal microscope. RESULTS: Significant CD4/FoxP3 Treg infiltrates were observed in tolerant donor-allograft skin samples. No graft infiltrating FoxP3 cells were observed in rejector, naïve, or skin from syngeneic CTA. In parallel experiments, mixed leukocyte reaction assays were performed to investigate the suppressor function of Treg cells. Splenocytes from tolerant, rejected, and naïve rats were sorted by flow cytometry for CD4/CD25 T cells. Treg demonstrated similar suppressive levels between the three groups. CONCLUSIONS: These data suggest that Treg may play an important role in maintenance of tolerance and promoting graft acceptance in long-term CTA acceptors and may explain the favorable outcomes observed in clinical CTA recipients.

In vivo observations of cell trafficking in allotransplanted vascularized skin flaps and conventional skin grafts.

The problem of allogeneic skin rejection is a major limitation to more widespread application of clinical composite tissue allotransplantation (CTA). Previous research examining skin rejection has mainly studied rejection of conventional skin grafts (CSG) using standard histological techniques. The aim of this study was to objectively assess if there were differences in the immune response to CSG and primarily vascularized skin in composite tissue allotransplants (SCTT) using in vivo techniques in order to gain new insights in to the immune response to skin allotransplants. CSG and SCTT were transplanted from standard Lewis (LEW) ad Wistar Furth (WF) to recipient transgenic green fluorescent Lewis rats (LEW-GFP). In vivo confocal microscopy was used to observe cell trafficking within skin of the transplants. In addition, immunohistochemical staining was performed on skin biopsies to reveal possible expression of class II major histocompatibility antigens. A difference was observed in the immune response to SCTT compared to CSG. SCTT had a greater density cellular infiltrate than CSG (p<0.03) that was focused more at the center of the transplant (p<0.05) than at the edges, likely due to the immediate vascularization of the skin. Recipient dendritic cells were only observed in rejecting SCTT, not CSG. Furthermore, dermal endothelial class II MHC expression was only observed in allogeneic SCTT. The immune response in both SCTT and CSG was focused on targets in the dermis, with infiltrating cells clustering around hair follicles (CSG and SCTT; p<0.01) and blood vessels (SCTT; p<0.01) in allogeneic transplants. This study suggests that there are significant differences between rejection of SCTT and CSG that may limit the relevance of much of the historical data on skin graft rejection when applied to composite tissue allotransplantation. Furthermore, the use of novel in vivo techniques identified characteristics of the immune response to allograft skin not previously described, which may be useful in directing future approaches to overcoming allograft skin rejection.

The effects of CD133-positive cells to a nonvascularized fasciocutaneous free graft in the rat model.

A fasciocutaneous free graft could prove the ideal free tissue graft to prevent contracture following adhesion of the graft to the recipient bed. However, for graft survival, vascular anastomosis involving complicated surgical techniques is necessary. In the present study, we focused on peripheral blood-derived CD133(+) cells, including the endothelial progenitor cell fraction. The purpose of this study is to investigate whether locally transplanted CD133(+) cells could increase revascularization and improve the quality of free and thick grafted tissue.On the abdomen of nude rats (F344/N Jcl rnu/rnu), a 5 x 2-cm fasciocutaneous graft was designed, which was grafted on to the back. After operation, rats received local injection of 1 x 10(5) human CD133(+) cells resuspended in 400 microL of phosphate-buffered saline (PBS) (CD133(+) group) or the same volume of PBS without cells (control group) (n = 10 in each group). In the CD133(+) group graft perfusion or graft survival was not improved significantly over controls. However, the capillary density increased markedly in the surrounding fascia and the dermis matured at an earlier stage in the CD133(+) group. In this study, we demonstrated that transplanted CD133(+) cells induced new blood vessel growth and improved dermis quality. Cell therapy with CD133(+) cells is already applied in a clinical setting because autologous cells can be used and therefore these procedures are not hampered by ethical concerns. CD133(+) cell transfer therefore has great potential as an adjunct therapy in the realm of microsurgery.

Immunologic responses in vascularized and nonvascularized skin allografts.

The skin is a protective interface between internal organs and the environment; it is the largest tissue of the human body. However, the skin does not serve merely as a physical barrier. It is also an active immune organ, traversed by a network of lymphatic and blood vessels. This immunologic structure contains immunologic cells such as T lymphocytes, Langerhans cells (LCs), dendritic cells, and keratinocytes. Langerhans cells represent the cutaneous counterpart of dendritic cells. LCs not only act as professional antigen-presenting cells to induce antigen-specific T cells for adaptive immune responses, but they also initiate a cascade of innate immune responses by antigenic stimulus such as transplant tissue. In transplantation immunology, either donor or recipient LCs fulfill an important mission in rejection or acceptance of donor tissue. Vascularized or nonvascularized skin allografts may create an immunologic response through different pathways. In both transplant models, skin diameter may change antigenic load, thereby determining rejection or acceptance response. This article discusses the effects of the cellular component in the skin immune system on immunologic responses of vascularized or nonvascularized skin allografts and describes the differences between the two immunologic cascades.

Reconstrucción auricular: experiencia de 24 años / Auricular reconstruction: 24 year experience

La oreja, debido a su situación expuesta en la cabeza, es un órgano susceptible de sufrir múltiples traumase incluso pérdida; así mismo, es asiento de neoplasias malignas, carcinomas y melanomas que igualmente finalizan en amputación. Fuera de estas razones, la ausencia congénita del pabellón auricular es la segunda malformación facial de origen congénito más frecuente después del labio y paladar fisurados. Es común por tanto para el cirujano plástico verse frente a pacientes que consultan solicitando la soluciona su deformidad. El presente artículo trata de señalar el camino a seguir dividido en cuatro etapas de acuerdo a los principios definidos por el Dr. Radford Tanzer y perfeccionados a través de los años por el Dr. Burt Brent, e ilustra el manejo de la reconstrucción en casos de microtia, amputación auricular y el uso de colgajos de fascia temporal en los casos en que la piel de la región mastoidea ha sido comprometida y no es posible usarla como elemento primario para la reconstrucción auricular (AU)

Due to its very exposed situation on both sides of the head, the auricle is very susceptible to suffer severe traumas, that will at the end cause its amputation; it’s too one of the most common sites to present malignant tumours, carcinoma and melanoma, that will also end in the surgical amputation of the ear. Congenital absence of the ear, microtia, is the second most common facial anomaly after cleft lip and palate. Not infrequently plastic surgeon confronts with patients seeking an answer for correction of their deformities. The present article tries to show a four steps method, according to the principles described first by Dr. Radford Tanzer and later improved by Dr. Burt Brent and illustrates how to manage the auricular reconstruction in cases of congenital microtia or traumatic loss of the auricle, as well as the use of the temporal fascia flap as a way to provide skin coverage, when the skin of the mastoid area has been compromised and it is not useful for auricular reconstruction (AU)

Effect of platelet glycoprotein IIb/IIIa receptor antagonist on survival of epigastric island flaps in rats with total venous occlusions.

We evaluated the effect of tirofiban hydrochloride on the survival of epigastric island flaps in rats that had had all the veins occluded. Male Wistar Albino rats were randomly assigned to control (treated with sterile saline) and experimental (treated with tirofiban hydrochloride 1 mg/kg intravenously) groups. An epigastric island skin flap 3x6 cm was raised in each rat. All veins that drained the flap were ligated to give total venous occlusion. Blood flow was recorded by laser Doppler preoperatively (baseline), immediately after the flap had been sutured back to its original position (acute) and on postoperative days 1 and 3. The degree of necrosis was evaluated on day 3. Mean percentage necrosis and minimum laser Doppler values were compared in the two groups. Total necrosis was evident on day 1 in the control group and on day 3 in the experimental group. Macroscopic evidence was confirmed by histopathological examination. There were appreciable differences in blood flow and in the necrotic area of the flap in the experimental group compared with the control group on both days 1 and 3. Tirofiban hydrochloride might be effective in this flap model.

A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts.

Composite tissue allotransplantation (CTA) is the new frontier in transplantation. More than 25 hand allograft transplants have been performed worldwide, and the feasibility has been well established. The classical experimental model of CTA involves rat orthotopic hindlimb transplantation, a time-consuming procedure associated with high mortality and morbidity. We describe a rat heterotopic osteomyocutaneous flap that serves as a nonfunctional CTA, allowing the study of tolerance induction to a highly antigenic vascularized allograft of bone, muscle, and skin while minimizing the morbidity and mortality of full hind limb transplantation. In the present studies, we explored whether establishing chimerism by nonmyeloablative conditioning would induce tolerance to CTA. When compared with the classic hind limb transplantation model, these results demonstrate that our heterotopic hind limb flap is less morbid and as an effective experimental model for the study of CTA tolerance.

Extracorporeal shock wave enhanced extended skin flap tissue survival via increase of topical blood perfusion and associated with suppression of tissue pro-inflammation.

OBJECTIVE: Distal skin flap ischemic necrosis is a significant challenge in reconstructive surgery. This study assessed whether extracorporeal shock wave (ESW) treatment rescues compromised flap tissue by enhancing tissue perfusion and is associated with suppression of inflammatory response. METHODS: This study used the dorsal skin random flap model in a rodent. Thirty-six male Sprague Dawley rats were divided into three groups. Group I, a control group, received no treatment. Group II was administrated 500 impulses of ESW treatment at 0.15 mJ/mm(2) as a single treatment immediately postoperatively. Group III received 500 impulses of ESW at 0.15 mJ/mm(2) applied immediately postoperatively and the day following surgery. Flap blood perfusion was detected by laser Doppler. Flap survival/necrosis area and histological staining of flap ischemia zone was performed on day 7 postoperatively. The tumor necrosis factor alpha, vascular endothelial growth factor, and proliferating cell nuclear antigen expression were evaluated with immunohistochemical staining. RESULTS: Experimental results indicated that the necrotic area of the flaps in Group II was significantly reduced compared with that in the control group (13 +/- 2.6% versus 42 +/- 5.7%, P < 0.01). There was small and insignificant reduction in the necrotic area in Group III compared with the controls. Flap tissue blood perfusion was significantly increased postoperatively in Group II. Histological staining indicated that ESW treatment substantially increased vascular endothelial growth factor and proliferating cell nuclear antigen expressions, reduced leukocyte infiltration, and suppression of tumor necrosis factor alpha expression in flap tissue ischemic zones in Group II compared with that in controls. CONCLUSION: Optimal dosage of ESW treatment has a positive effect in rescuing ischemic zone of flap by increasing tissue perfusion and is associated with suppressing inflammatory response.

Pro-inflammatory cytokines in elective flap surgery.

BACKGROUND: Surgical trauma releases inflammatory mediators such as pro-inflammatory cytokines. In this prospective, controlled, randomized trial we investigated the release of pro-inflammatory cytokines and monocyte/macrophage activation in patients scheduled for breast reconstruction after mastectomy. Patients were allocated to one of three surgical procedures, differing in complexity and in the need for implants used for reconstruction. METHODS: Thirty mastectomized women underwent delayed breast reconstruction with the lateral thoracodorsal flap (LTD), the latissimus dorsi flap (LD), or the pedicled transverse rectus abdominis muscle flap (TRAM). Blood samples for TNF, IL-6, IL-8, neopterin, C-reactive protein (CRP), and leukocyte determination were drawn pre-operatively, 24 h, and 2 weeks post-operatively. RESULTS: All groups had significantly elevated IL-6 levels 24 h after surgery. The levels were significantly higher in the TRAM group compared to the LTD and LD groups. IL-8 levels were increased in all groups 2 weeks after surgery (P < 0.05), the LTD (83 pg/mL) and LD (84 pg/mL) group having higher mean IL-8 levels than the TRAM patients (48 pg/mL) (ns). TNF and leukocyte counts were within the normal range. CRP levels were elevated in all groups one day after surgery (P < 0.05). CONCLUSION: Flap procedures for breast reconstruction stimulate the pro-inflammatory response. IL-6 levels were highest in patients with TRAM operations, being the most extensive procedure studied, whereas the highest IL-8 levels were seen in women with a saline filled silicone implant suggesting immunomodulation by foreign material. Although all three investigated procedures are major operations in the field of plastic surgery, according to the inflammatory response to trauma they should be regarded as minor procedures.

Strategies to develop chimerism in vascularized skin allografts across MHC barrier.

In this study, we investigated the effects of 7-day-protocols of alphabeta-T-cell receptor monoclonal antibody (alphabeta-TCRmAb), cyclosporine A (CsA), and tacrolimus (FK-506) immunosuppressive monotherapies, and their combinations on the survival of vascularized skin allografts (VSA). Forty-two transplantations of VSA across a strong MHC barrier were performed between ACI (RT1a) donors and Lewis (RT1(l)) recipients in seven groups. Isograft and allograft rejection controls received no treatment. Treatment groups received a 7-day protocol of alphabeta-TCRmAb, CsA, or FK-506 monotherapy, or a combination of alphabeta-TCRmAb/CsA and alphabeta-TCRmAb/FK-506. VSA transplants were evaluated on a daily basis. Donor-specific chimerism was determined by flow cytometry (FC). The combined protocols of alphabeta-TCRmAb/FK-506 and alphabeta-TCRmAb/CsA significantly prolonged VSA survivals compared to monotherapy groups ( P < 0.005). FC analysis revealed 15.82% of donor-specific chimerism on day 7 under the alphabeta-TCRmAb/CsA protocol and a gradual chimerism decline on day 63 posttransplant. The significant extension of VSA survival achieved under 7-day protocols of combined therapies was directly associated with the presence of donor-specific chimerism.

Immunology of skin transplantation.

Untreated viable allogeneic skin is highly immunogenic. Epidermal Langerhans migrate after transplantation out of the donor skin into the lymph node of the recipient where they can activate T cells capable to mediate rejection. Allogeneic skin is used as a temporary coverage of burn wounds, often in combination with autologous skin grafts. Several methods to pretreat the allogeneic skin have been used to delay the rejection process. Processing of allogeneic skin in 85% glycerol results in a non-viable skin with a well-preserved structure. Experiments in a full thickness porcine wound model showed that rejection of glycerol treated allogeneic skin grafts was up to six days delayed. Viable, untreated allogeneic skin grafts were rejected predominantly by CD8 positive T cells whereas in the glycerol treated grafts the influx of host cells was lower and the majority of the cells were macrophages. The outgrowth of the autologous skin grafts underneath glycerol treated allogeneic skin was three days earlier completed when compared to grafts in combination with untreated allogeneic skin. Thus, by processing the allogeneic skin into 85% glycerol, the direct route to induce graft rejection is blocked since the Langerhans cells are non-viable. The glycerol-preserved skin grafts are finally rejected via an indirect route mediated by macrophages; this process is less disturbing for the outgrowth of autologous cells.

Anti-TGFbeta1 antibody for modulation of expression of endogenous transforming growth factor beta 1 to prevent fibrosis after plastic surgery in rats.

The cytokine transforming growth factor beta 1 (TGFbeta1) substantially influences synthesis of extracellular matrix, fibrosis, and neoangiogenesis during wound healing in a dose dependent manner. We carried out experiments in rats to measure the degree of reduction of disorders of wound healing and fibrosis produced by inhibition of endogenous TGFbeta1 by polyclonal antibodies (poAB). A free myocutaneous gracilis flap was transplanted from the groin to the neck region in 30 Wistar rats (300-450 g body weight). In 15 animals intraoperatively and daily from days 3 to 7 postoperatively, 1 microg anti-TGFbeta1 poAB in 500 microl of phosphate buffered saline (PBS) were injected into the neck region. Fifteen animals served as controls. On postoperative days 3, 4, 5, 7, 14, and 28 the expression of endogenous TGFbeta1 in cytoplasm was analysed by immunohistochemistry (ABC-POX; AEC), in situ hybridisation of TGFbeta1-mRNA, and Sirus Red staining of collagen matrix; it was quantified using labelling indices. Neutralisation of the TGFbeta1 activity by specific poAB resulted in inhibition of the cytoplasmatic expression compared with untreated animals. In the transition area between grafted tissue and graft bed, a significant reduction of TGFbeta1 expression (mean (S.D.) 34.7 (6.5)) was found from day 5 in the group treated with anti-TGFbeta1 poAB compared with the control group (mean (S.D.) 48.1 (6.6)) (P <0.03). Up to day 14 the endogenous expression of TGFbeta1 (mean (S.D.) 30.0 (2.8)) was reduced after the application of TGFbeta1 poAB compared with the control group (mean (S.D.) 44.0 (12.3)). Sirus Red staining indicated a more complex packed structure and generally more prominent collagen types I-IV fibres in untreated animals than in animals that were given anti-TGFbeta1 poAB. Expression of TGFbeta-mRNA by in situ hybridisation was reduced in fibroblasts in animals that were given anti-TGFbeta1 poAB. The results indicate that anti-TGFbeta1 might improve the healing of free flaps in the graft beds of patients who are prone to excessive fibrosis.