Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner.

BACKGROUND AIMS: Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS: Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS: Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1ß, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION: We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.

Botulinum toxin A and B raise blood flow and increase survival of critically ischemic skin flaps.

BACKGROUND: Botulinum toxin (BTX) A and B are commonly used for aesthetic indications and in neuromuscular disorders. New concepts seek to prove efficacy of BTX for critical tissue perfusion. Our aim was to evaluate BTX A and B in a mouse model of critical flap ischemia for preoperative and intraoperative application. METHODS: BTX A and B were applied on the vascular pedicle of an axial pattern flap in mice preoperatively or intraoperatively. Blood flow, tissue oxygenation, tissue metabolism, flap necrosis rate, apoptosis assay, and RhoA and eNOS expression were endpoints. RESULTS: Blood-flow measurements 1 d after the flap operation revealed a significant reduction to 53% in the control group, while flow was maintained or increased in all BTX groups (103%-129%). Over 5 d all BTX groups showed significant increase in blood flow to 166-187% (P < 0.01). Microdialysis revealed an increase of glucose and reduced lactate/pyruvate ratio and glycerol levels in the flap tissue of all BTX groups. This resulted in significantly improved tissue survival in all BTX groups compared with the control group (62% ± 10%; all P < 0.01): BTX A preconditioning (84% ± 5%), BTX A application intraoperatively (88% ± 4%), BTX B preconditioning (91% ± 4%), and intraoperative BTX B treatment (92% ± 5%). This was confirmed by TUNEL assay. Immunofluorescence demonstrated RhoA and eNOS expression in BTX groups. All BTX applications were similarly effective, despite pharmacologic dissimilarities and different timing. CONCLUSIONS: In conclusion, we were able to show on a vascular, tissue, cell, and molecular level that BTX injection to the feeding arteries supports flap survival through ameliorated blood flow and oxygen delivery.

Distinct microhemodynamic efficacy of arteriogenesis and angiogenesis in critically ischemic skin flaps.

Angiogenesis and arteriogenesis are regenerative vascular mechanisms dedicated to cope with critical ischemia after the interruption of the anatomical axial blood supply. The aim of the present study was to visualize, quantify and monitor the orchestration of these mechanisms and their microhemodynamic efficacy. A murine skin flap model was used that allowed for repetitive investigation of identical vascular structures by intravital microscopy. In the conduit arterioles, diameter and relative length increased to 133 ± 20% and 260 ± 80% over 7 days, respectively (both P<0.01), which reduced vascular resistance in this segment to 82 ± 35%. After 1 week, a peak in accumulation of activated leukocytes could be observed in the postcapillary venules (P<0.01) without relevant hemodynamic changes. Thereafter, the arteriolar remodeling was replaced by angiogenesis. Functional capillary density was increased to 141 ± 10% (P<0.01) and capillary diameter to 123 ± 6% (P<0.01) after 14 days. Both mechanisms of vascular regeneration were associated with increases in the capillary perfusion index, to 194 ± 42% (P<0.05) after 7 days and 366 ± 21% after 14 days (P>0.01). Immunohistochemical analysis revealed a correlation of arteriogenesis with eNOS upregulation and of angiogenesis with VEGF upregulation in the corresponding vessels. In conclusion, arteriogenesis was the initial regenerative mechanism leading to arteriolar remodeling, reduction in vascular resistance, and increase in capillary perfusion over the first 7 days. Thereafter, capillary perfusion was improved by angiogenesis in terms of an increase in functional capillary density.

Paracrine effects of mesenchymal stem cells enhance vascular regeneration in ischemic murine skin.

New theories on the regeneration of ischemic vasculature have emerged indicating a pivotal role of adult stem cells. The aim of this study was to investigate homing and hemodynamic effects of circulating bone marrow-derived mesenchymal stem cells (MSCs) in a critically ischemic murine skin flap model. Bone marrow-derived mesenchymal stem cells (Lin(-)CD105(+)) were harvested from GFP(+)-donor mice and transferred to wildtype C57BL/6 mice. Animals receiving GFP(+)-fibroblasts served as a control group. Laser scanning confocal microscopy and intravital fluorescence microscopy were used for morphological analysis, monitoring and quantitative assessment of the stem cell homing and microhemodynamics over two weeks. Immunohistochemical staining was performed for GFP, eNOS, iNOS, VEGF. Tissue viability was analyzed by TUNEL-assay. We were able to visualize perivascular homing of MSCs in vivo. After 4 days, MSCs aligned along the vascular wall without undergoing endothelial or smooth muscle cell differentiation during the observation period. The gradual increase in arterial vascular resistance observed in the control group was abolished after MSC administration (P<0.01). At capillary level, a strong angiogenic response was found from day 7 onwards. Functional capillary density was raised in the MSC group to 197% compared to 132% in the control group (P<0.01). Paracrine expression of VEGF and iNOS, but not eNOS could be shown in the MSC group but not in the controls. In conclusion, we demonstrated that circulating bone marrow-derived MSCs home to perivascular sites in critically ischemic tissue, exhibits paracrine function and augment microhemodynamics. These effects were mediated through arteriogenesis and angiogenesis, which contributed to vascular regeneration.

The choice of anesthesia influences oxidative energy metabolism and tissue survival in critically ischemic murine skin.

In surgical animal studies anesthesia is used regularly. Several reports in the literature demonstrate respiratory and cardiovascular side effects of anesthesiologic agents. The aim of this study was to compare two frequently used anesthesia cocktails (ketamine/xylazine [KX] versus medetomidine/climazolam/fentanyl [MCF]) in skin flap mouse models. Systemic blood values, local metabolic parameters, and surgical outcome should be analyzed in critical ischemic skin flap models. Systemic hypoxia was found in the animals undergoing KX anesthesia compared with normoxia in the MCF group (sO(2): 89.2% +/- 2.4% versus 98.5% +/- 1.2%, P < 0.01). Analysis of tissue metabolism revealed impaired anaerobic oxygen metabolism and increased cellular damage in critical ischemic flap tissue under KX anesthesia (lactate/pyruvate ratio: KX 349.86 +/- 282.38 versus MCF 64.53 +/- 18.63; P < 0.01 and glycerol: KX 333.50 +/- 83.91 micromol/L versus MCF 195.83 +/- 29.49 micromol/L; P < 0.01). After 6 d, different rates of flap tissue necrosis could be detected (MCF 57% +/- 6% versus KX 68% +/- 6%, P < 0.01). In summary we want to point out that the type of anesthesia, the animal model and the goal of the study have to be well correlated. Comparing the effects of KX and MCF anesthesia in mice on surgical outcome was a novel aspect of our study.

Hemoglobin vesicles improve wound healing and tissue survival in critically ischemic skin in mice.

Local hypoxia, as due to trauma, surgery, or arterial occlusive disease, may severely jeopardize the survival of the affected tissue and its wound-healing capacity. Initially developed to replace blood transfusions, artificial oxygen carriers have emerged as oxygen therapeutics in such conditions. The aim of this study was to target primary wound healing and survival in critically ischemic skin by the systemic application of left-shifted liposomal hemoglobin vesicles (HbVs). This was tested in bilateral, cranially based dorsal skin flaps in mice treated with a HbV solution with an oxygen affinity that was increased to a P(50) (partial oxygen tension at which the hemoglobin becomes 50% saturated with oxygen) of 9 mmHg. Twenty percent of the total blood volume of the HbV solution was injected immediately and 24 h after surgery. On the first postoperative day, oxygen saturation in the critically ischemic middle flap portions was increased from 23% (untreated control) to 39% in the HbV-treated animals (P < 0.05). Six days postoperatively, flap tissue survival was increased from 33% (control) to 57% (P < 0.01) and primary healing of the ischemic wound margins from 6.6 to 12.7 mm (P < 0.05) after HbV injection. In addition, higher capillary counts and endothelial nitric oxide synthase expression (both P < 0.01) were found in the immunostained flap tissue. We conclude that left-shifted HbVs may ameliorate the survival and primary wound healing in critically ischemic skin, possibly mediated by endothelial nitric oxide synthase-induced neovascularization.

Ischemia-induced up-regulation of heme oxygenase-1 protects from apoptotic cell death and tissue necrosis.

BACKGROUND: Tissues are endowed with protective mechanisms to counteract chronic ischemia. Previous studies have demonstrated that endogenous heme oxygenase (HO)-1 may protect parenchymal tissue from inflammation- and reoxygenation-induced injury. Nothing is known, however, on whether endogenous HO-1 also plays a role in chronic ischemia to protect from development of tissue necrosis. The aim of this study is, therefore, to evaluate in vivo whether endogenous HO-1 exerts protection on chronically ischemic musculocutaneous tissue, and whether this protection is mediated by an attenuation of the microcirculatory dysfunction. MATERIALS AND METHODS: In C57BL/6-mice, a chronically ischemic flap was elevated and fixed into a dorsal skinfold chamber. In a second group, tin-protoporphyrin-IX was administrated to competitively block the action of HO-1. Animals without flap elevation served as controls. With the use of intravital fluorescence microscopy, microcirculation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day observation period. The time course of HO-1 expression was determined by Western blotting. RESULTS: Chronic ischemia induced an increase of HO-1 expression, particularly at day 1 and 3. This was associated with arteriolar dilation and hyperperfusion, which was capable of maintaining an adequate capillary perfusion density in the critically perfused central part of the flap, demarcating the distal necrosis. Inhibition of endogenous HO-1 by tin-protoporphyrin-IX completely abrogated arteriolar dilation (44.6 +/- 6.2 microm versus untreated flaps: 71.3 +/- 7.3 microm; P < 0.05) and hyperperfusion (3.13 +/- 1.29 nL/s versus 8.55 +/- 3.56 nL/s; P < 0.05). This resulted in a dramatic decrease of functional capillary density (16 +/- 16 cm/cm(2)versus 84 +/- 31 cm/cm(2); P < 0.05) and a significant increase of apoptotic cell death (585 +/- 51 cells/mm(2)versus 365 +/- 53 cells/mm(2); P < 0.05), and tissue necrosis (73% +/- 5% versus 51% +/- 5%; P < 0.001). CONCLUSION: Thus, our results suggest that chronic ischemia-induced endogenous HO-1 protects ischemically endangered tissue, probably by the vasodilatory action of the HO-1-associated carbon monoxide.

Outcome analysis of breast reconstruction with free transverse rectus abdominis musculocutaneous (TRAM) flaps.

QUESTIONS UNDER STUDY: With the reduction in breast cancer mortality in recent years the aesthetic outcome after treatment has gained increasing attention. The aim of this study was to assess the outcome quality of our single institution concept of free TRAM flap breast reconstruction with the aim of providing data to assist the patient's decision-making when breast reconstruction is an option. PRINCIPLES/METHODS: Thirty-two consecutive patients receiving immediate (n = 14) or delayed (n = 18) breast reconstruction with free transverse rectus abdominis musculocutaneous (TRAM) flaps were included. The selection of patients was based on their own wish and the availability of abdominal tissue, without excluding patients at risk for wound healing complications. Patient data were assessed prospectively and the aesthetic outcome was rated after the final result had been achieved. RESULTS: Ten patients sustained wound healing complications (4 of 9 smokers, 8 of 16 patients with a BMI over 25 kg/m2), 8 of them requiring revisional surgery. An average of 1.06 corrective procedures were performed per patient. The aesthetic outcome was judged to be good by 20 patients, fair by 5 and poor by 1 patient who sustained severe tissue loss. CONCLUSIONS: Our results revealed that a large measure of satisfaction is achievable from breast reconstruction with free TRAM flaps, in spite of the invasive nature of the procedure and the inclusion of patients at risk for wound healing complications. These data may be used in the decision-making process by patients eligible for breast reconstruction after mastectomy.

Aging is associated with an increased susceptibility to ischaemic necrosis due to microvascular perfusion failure but not a reduction in ischaemic tolerance.

In the present study in a murine model of chronic ischaemia, we analysed: (i) whether aging was associated with an increased susceptibility to ischaemic necrosis, and (ii) whether this was based on microvascular dysfunction or reduced ischaemic tolerance. An ischaemic pedicled skin flap was created in the ear of homozygous hairless mice. The animals were assigned to three age groups, including adolescent (2+/-1 months), adult (10+/-2 months) and senescent (19+/-3 months). Microvascular perfusion of the ischaemic flap was assessed over 5 days by intravital microscopy, evaluating FCD (functional capillary density), capillary dilation response and the area of tissue necrosis. Expression of the stress-protein HO (haem oxygenase)-1 was determined by immunohistochemistry and Western blotting. Induction of chronic ischaemia stimulated a significant expression of HO-1 without a significant difference between the three age groups. This was associated with capillary dilation, which, however, was more pronounced in adolescent (10.5+/-2.8 microm compared with 3.95+/-0.79 microm at baseline) and adult (12.1+/-3.1 microm compared with 3.36+/-0.45 microm at baseline) animals compared with senescent animals (8.5+/-1.7 microm compared with 3.28+/-0.69 microm at baseline; P value not significant). In senescent animals, flap creation further resulted in complete cessation of capillary flow in the distal area of the flap (FCD, 0+/-0 cm/cm(2)), whereas adult (11.9+/-13.5 cm/cm(2)) and, in particular, adolescent animals (58.4+/-33.6 cm/cm(2); P<0.05) were capable of maintaining residual capillary perfusion. The age-associated microcirculatory dysfunction resulted in a significantly increased flap necrosis of 49+/-8% (P<0.05) and 42+/-8% (P<0.05) in senescent and adult animals respectively, compared with 31+/-6% in adolescent mice. Of interest, functional inhibition of HO-1 by SnPP-IX (tin protoporphyrin-IX) in adolescent mice abrogated capillary dilation, decreased functional capillary density and aggravated tissue necrosis comparably with that observed in senescent mice. Thus aging is associated with an increased susceptibility to tissue necrosis, which is due to a loss of vascular reactivity to endogenous HO-1 expression, rather than a reduction in ischaemic tolerance.

Rapid restoration of microcirculatory blood flow with hyperviscous and hyperoncotic solutions lowers the transfusion trigger in resuscitation from hemorrhagic shock.

Resuscitation from hemorrhagic shock relies on fluid retransfusion. However, the optimal properties of the fluid have not been established. The aim of the present study was to test the influence of the concentration of hydroxyethyl starch (HES) solution on plasma viscosity and colloid osmotic pressure (COP), systemic and microcirculatory recovery, and oxygen delivery and consumption after resuscitation, which were assessed in the hamster chamber window preparation by intravital microscopy. Awake hamsters were subjected to 50% hemorrhage and were resuscitated with 25% of the estimated blood volume with 5%, 10%, or 20% HES solution. The increase in concentration led to an increase in COP (from 20 to 70 and 194 mmHg) and viscosity (from 1.7 to 3.8 and 14.4 cP). Cardiac index and microcirculatory and metabolic recovery were improved with HES 10% and 20% when compared with 5% HES. Oxygen delivery and consumption in the dorsal skinfold chamber was more than doubled with HES 10% and 20% when compared with HES 5%. This was attributed to the beneficial effect of restored or increased plasma COP and plasma viscosity as obtained with HES 10% and 20%, leading to improved microcirculatory blood flow values early in the resuscitation period. The increase in COP led to an increase in blood volume as shown by a reduction in hematocrit. Mean arterial pressure was significantly improved in animals receiving 10% and 20% solutions. In conclusion, the present results show that the increase in the concentration of HES, leading to hyperoncotic and hyperviscous solutions, is beneficial for resuscitation from hemorrhagic shock because normalization of COP and viscosity led to a rapid recovery of microcirculatory parameters.

Early resuscitation with polymerized bovine hemoglobin reverses acidosis, but not peripheral tissue oxygenation, in a severe hamster shock model.

Awake hamsters equipped with the dorsal window chamber preparation were subjected to hemorrhage of 50% of the estimated blood volume. Initial resuscitation (25% of estimated blood volume) with polymerized bovine hemoglobin (PBH) or 10% hydroxyethyl starch (HES) occurred in concert with an equivolumetric bleeding to simulate the early, prehospital setting (exchange transfusion). Resuscitation (25% of estimated blood volume) without bleeding was performed with PBH, HES, or autologous red blood cells (HES-RBCs). Peripheral microcirculation, tissue oxygenation, and systemic hemodynamic and blood gas parameters were assessed. After exchange transfusion, base deficit was -8.6 +/- 3.7 mmol/L (PBH) and -5.1 +/- 5.3 mmol/L (HES) (not significant). Functional capillary density was 17% +/- 6% of baseline (PBH) and 31% +/- 11% (HES) (P < 0.05) and arteriolar diameter 73% +/- 3% of baseline (PBH) and 90% + 5% (HES) (P < 0.01). At the end, hemoglobin levels were 3.7 +/- 0.3 g/dL with HES, 8.2 +/- 0.6 g/dL with PBH, and 10.4 +/- 0.8 g/dL with HES-RBCs (P < 0.01 HES vs. PBH and HES-RBCs, P < 0.05 PBH vs. HES-RBCs). Base excess was restored to baseline with PBH and HES-RBCs, but not with HES (P < 0.05). Functional capillary density was 46% +/- 5% of baseline (PBH), 62% + 20% (HES-RBCs), and 36% +/- 19% (HES) (P < 0.01 HES-RBCs vs. HES). Peripheral oxygen delivery and consumption was highest with HES-RBCs, followed by PBH (P < 0.05 HES-RBCs vs. PBH, P < 0.01 HES-RBCs and PBH vs. HES). In conclusion, the PBH led to a correction of base deficit comparable to blood transfusion. However, oxygenation of the peripheral tissue was inferior with PBH. This was attributed to its negative impact on the peripheral microcirculation caused by arteriolar vasoconstriction.

Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin.

Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.

Resuscitation from hemorrhagic shock with MalPEG-albumin: comparison with MalPEG-hemoglobin.

Our aim was to determine the efficacy of polyethylene glycol-conjugated human albumin (MalPEG-Alb) in restoring circulatory volume after 1 h of hemorrhagic shock. Experiments were performed in the awake condition in the hamster skin fold preparation. Microhemodynamic parameters and tissue Po2 were assessed with intravital microscopy and the use of the phosphorescence quenching technique. One hour after shock induction by withdrawal of 50% of the blood volume, animals were resuscitated with MalPEG-Alb (n = 6). Systemic and microhemodynamic parameters following resuscitation were identical to those obtained with the same protocol using MalPEG-Hb (1). However, parameters related to microvascular oxygen distribution were significantly lower in the MalPEG-Alb group compared with the previous data from the MalPEG-Hb group in that tissue oxygen partial pressure was 5 +/- 2 mmHg (vs. 8 +/- 3 mmHg, P < 0.05), oxygen delivery was reduced to 60 +/- 27% (P < 0.05), and oxygen consumption was reduced to 69 +/- 28% (P < 0.05). Both molecules were matched in composition (4.2 g/dL) and surface chemistry. MalPEG-Alb colloid osmotic pressure was 37 mmHg (vs. 49 mmHg for MalPEG-Hb), and viscosity was 2.7 cP (vs. 2.5 cP for MalPEG-Hb). The present results show that both solutions are efficacious plasma expanders and that the hemoglobin-based solution provides improved oxygen distribution and tissue Po2 in the hamster chamber model.

Improving microcirculation is more effective than substitution of red blood cells to correct metabolic disorder in experimental hemorrhagic shock.

Microcirculatory perfusion deficits and impaired tissue oxygenation in nonvital organs frequently occur after hemorrhage and they contribute to potentially lethal complications. The aim of this study was to test the influence of colloid osmotic pressure, viscosity, and red blood cell (RBC) content of the resuscitative fluid on metabolic disorder, perfusion, and oxygenation in peripheral tissues. Awake hamsters were subjected to hemorrhage of 50% and were resuscitated with 25% of blood volume with solutions containing 6% pegylated bovine albumin only (PEG-BSA 0) and 6% PEG-BSA mixed with autologous RBCs to reach 4 g/dL (PEG-BSA 4) and 8 g/dL (PEG-BSA 8) of hemoglobin. PEG-BSA had a viscosity of 4.2 cP and a COP of 116 mmHg. Microhemodynamics and tissue pO2 were assessed in the hamster chamber window preparation with intravital microscopy. Arterial base excess tended to be lower than baseline for PEG-BSA 0 and PEG-BSA 4 (ns), whereas base deficit remained significantly decreased for PEG-BSA 8 (P<0.05 vs. baseline). Oxygen extraction was 91% +/- 2% of the oxygen delivery for PEG-BSA 0 compared with 85% +/- 2% for PEG-BSA 8 (P<0.05). Functional capillary density was 61%, 47%, and 45% for PEG-BSA 0 (P<0.05 vs. other groups), PEG-BSA 4 and PEG-BSA 8, respectively. We conclude that arterial base excess and oxygen extraction ratio in the tissue was better restored if a higher fraction of PEG-BSA and less RBCs were infused. This was attributed to a more homogeneous distribution of oxygen, as reflected by functional capillary density. Our results suggest that the transfusion trigger in hemorrhagic shock may be shifted toward lower hemoglobin concentrations if highly viscous and oncotic solutions are used.

Four years follow-up after clavicle reconstruction in a child: a case report.

Clavicle reconstruction is a rare operation. In most cases a mid-shaft defect of the clavicle is bridged by using different grafting techniques or musculo-osteous flaps. In some clinical situations where reconstruction is not a suitable option claviculectomy as a salvation procedure has proven to be an acceptable solution. In the paediatric population the challenge of both the cosmetic and the functional result attempting reconstruction of large bone defects is of higher demand. To our knowledge, this is the first case of a successful clavicle reconstruction with a sufficient follow-up using a free vascularised fibula graft in a child. This case provides a technique description, considerations in the paediatric population, an overview of other techniques used, and a long-term follow-up.

Perioperative risk factors for haematoma after breast augmentation.

Postoperative bleeding is the most frequent early complication after breast augmentation. The aim of this retrospective analysis was to assess possible risk factors originating in the perioperative management. All primary, bilateral breast augmentation procedures including augmentation mastopexy performed over an 8-year period were reviewed. Nine cases of postoperative bleeding could be identified in the 132 patients included in the study (6.8%). Univariate statistical analysis revealed age (p < 0.01), the amount of administered fluid intraoperatively (p < 0.05) and within the first 24 hours from the onset of anaesthesia (p < 0.01), the use of colloids (p < 0.01), total intravenous anaesthesia (p < 0.05), and systolic hypotension during the last 30 minutes of the operation (p < 0.05) as risk factors, whereas none of them could be identified as independent risk factor in multivariate analysis. In conclusion, this study was able to identify risk factors originating in the perioperative management that may lead to haematoma formation after breast augmentation.

One hundred fascia-sparing myocutaneous rectus abdominis flaps: An update.

Major efforts have been undertaken to reduce donor-site morbidity after abdominal flaps, which eventually culminated in the introduction of the deep inferior epigastric perforator (DIEP) flap. However, due to anatomical variations (absence of dominant perforators) and the risk of ischaemic complications, the selection of patients qualifying for a DIEP flap is limited. Furthermore, DIEP flaps can only be used as free flaps. We present our long-term experience with a dissection technique of rectus abdominis myocutaneous (RAM) flaps that was developed to circumvent these drawbacks. The dissection is characterised by preventing to sacrifice any perforators nourishing the flap and by fully preserving the anterior rectus sheath, but not the muscle. The study comprises a consecutive series of prospectively assessed patients, treated between February 2000 and April 2008. A total of 100 fascia-sparing RAM flaps were operated on 97 patients (age 22-84 years, median 64 years). Free flaps were mainly used for breast reconstruction (47 flaps/24 patients), and cranially (34) or caudally (19) pedicled flaps for soft-tissue coverage after sternectomy, urogenital tumour resection or rectum amputation. Eighty patients had a total of 213 risk factors, such as cardiovascular diseases, obesity, hyperlipidaemia, diabetes mellitus, smoking or steroid medication. Partial tissue loss (skin or fat necrosis) occurred in 13 flaps, out of which seven required surgical revision. The ischaemic complications were evenly distributed between the patient subsets. At a follow-up of 2-89 months (median 20 months), one patient showed a flap harvest-related abdominal bulge after bilateral-free transverse rectus abdominis myocutaneous (TRAM) flap. We conclude that the present dissection technique provides maximal perforator-related perfusion and minimal donor-site morbidity even in pedicled flaps and high-risk patients. In free flaps, it may, therefore, be recommended as an alternative to the DIEP flap.

Aesthetic outcome and oncological safety of nipple-areola complex replantation after mastectomy and immediate breast reconstruction.

Immediate breast reconstruction (IBR) has become an established procedure for women necessitating mastectomy. Traditionally, the nipple-areola complex (NAC) is resected during this procedure. The NAC, in turn, is a principal factor determining aesthetic outcome after breast reconstruction, and due to its particular texture and shape, a natural-looking NAC can barely be reconstructed with other tissues. The aim of this study was to assess the oncological safety as well as morbidity and aesthetic outcome after replantation of the NAC some days after IBR. Retrospective analysis of 85 patients receiving 88 mastectomies and IBR between 1998 and 2007 was conducted. NAC (n=29) or the nipple alone (n=23) were replanted 7 days (median, range 2-10 days) after IBR in 49 patients, provided the subareolar tissue was histologically negative for tumour infiltration. Local recurrence rate was assessed after 49 months (median, range 6-120 months). Aesthetic outcome was evaluated by clinical assessment during routine follow-up at least 12 months after the last intervention. Malignant involvement of the subareolar tissue was found in eight cases (9.1%). Patients qualifying for NAC replantation were in stage 0 in 29%, stage I in 15%, stage IIa in 31%, stage IIb in 17% and stage III in 8%. Total or partial necrosis occurred in 69% and 26% if the entire NAC or only the nipple were replanted, respectively (P<0.01). Depigmentation was seen in 52% and corrective surgery was done in 11 out of 52 NAC or nipple replantations. Local recurrence and isolated regional lymph node metastasis were observed in one single case each. Another 5.8% of the patients showed distant metastases. We conclude that the replantation of the NAC in IBR is oncologically safe, provided the subareolar tissue is free of tumour. However, the long-term aesthetic outcome of NAC replantation is not satisfying, which advocates replanting the nipple alone.

Vascularised fibula grafts for early tibia reconstruction in infants with congenital pseudarthrosis.

Congenital pseudarthrosis of the tibia (CPT) is caused by an ill-defined, segmental disturbance of periosteal bone formation leading to spontaneous bowing, followed by fracture and subsequent pseudarthrosis in the first 2 years of life. The results of conventional treatment modalities (e.g., bracing, internal and external fixation and bone grafting) are associated with high failure rates in terms of persisting pseudarthrosis, malunion and impaired growth. As a more promising alternative, a more aggressive approach, including wide resection of the affected bone, reconstruction with free vascularised fibula grafts from the healthy contralateral leg and stable external fixation at a very early stage has been suggested. Between 1995 and 2007, 10 children (age 12-31 months, median 20 months) suffering from CPT were treated at our institutions according to this principle. Two patients were treated before a fracture had occurred. The length of the fibula graft was 7-9cm. End-to-end anastomoses were performed at the level of the distal tibia stump. The follow-up was 80 months (median, range 12 months to 12 years). Radiologic examination at 6 weeks postoperatively showed normal bone density and structure of the transplanted fibula in all cases and osseous consolidation at 19 of the 20 graft/tibia junctions. One nonunion was sucessfully treated with bone grafting and plate osteosynthesis. Pin-tract infection occurred in three patients. Five children sustained graft fractures that were successfully treated with internal or external fixation. Two patients developed diminished growth of the affected limb or foot; all others had equal limb length and shoe size. At long-term follow-up, tibialisation of the transplant had occurred, and normal gait and physical activities were possible in all children. We conclude that in spite of a relatively high complication rate and the reluctance to perform free flap surgery in infants at this young age, the present concept may successfully prevent the imminent severe sequelae associated with CPT.