Establishment of a resazurin-based aortic valve tissue viability assay for dynamic culture in a microphysiological system.

BACKGROUND/AIM: Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS: A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm2) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50-300µM), incubation times and tissue masses and was consequently adapted. RESULTS: Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9-13.4µl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU)-ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300µM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS: Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

HELP-(Heparin-induced Extracorporeal LDL Precipitation)-apheresis in heart recipients with cardiac allograft vasculopathy and concomitant hypercholesterolemia: Influence of long-term treatment on the microcirculation.

Hyperlipidemic heart transplant patients who develop cardiac allograft vasculopathy (CAV) benefit from HELP-apheresis (Heparin-induced Extracorporeal LDL Precipitation) which enables drastic lowering of plasma low-density lipoprotein, lipoprotein (a), and fibrinogen. There is evidence that HELP-apheresis also improves microcirculation by an immediate improvement of impaired endothelial-dependent vasodilatation and additive hemorheological effects.Therefore, cutaneous microcirculation was examined before, during, and after the first HELP-apheresis in eight hyperlipidemic cardiac transplant recipients with CAV. To study the long-term effect the intravital microscopy was repeated after three and 12 months of weekly apheresis treatment.In CAV patients the baseline mean erythrocyte velocity was pathologically reduced with 0.13±0.07 mm/s. During the first HELP-apheresis the erythrocyte velocity increased significantly (p = 0.0001) and remained increased until the end of the HELP procedure (p < 0.05). After three months of weekly apheresis treatment a decrease of temporary flow stops in the capillaries with a progressive homogenization (concordance) of the cutaneous microcirculation was observed. After one year of weekly treatment a markedly increase in mean erythrocyte velocity under resting conditions occurred. In addition, a reactive post-ischemic hyperemia could be established for the first time.Even the first single HELP-apheresis resulted in a significant improvement of the cutaneous microcirculation. The long-term treatment of these patients resulted in a marked improvement of the cutaneous microcirculation with the tendency to a normalization of the regulation of the capillary perfusion.

Characterization of thoracal fat depots - expression of adipokines and remodeling factors and impact of adipocyte conditioned media in fibroblast scratch assays.

Adipose tissue is not only a connective tissue but also an endocrine organ secreting adipokines like Leptin and Adiponectin, lipokines such as palmitoileic acid and extracellular vesicles. These factors and the expression of matrix remodeling enzymes impact surrounding tissues via paracrine effects. The expression of selected secretion factors and the effect of adipocyte conditioned media from four thoracal adipose tissue origins - subcutaneous, perivascular, pericardial and epicardial adipose tissues - in a fibroblast proliferation/wound healing scratch assay model were investigated. Results were compared directly and according to the type 2 diabetic mellitus (T2DM) status of the patients the tissues are originated from. Adipocyte conditioned media from non-diabetic patients resulted in a significant higher scratch closure rate compared to the media with T2DM background. Linoleic acid incubation in scratch assay resulted in a reduced scratch closure rate. Leptin, Adiponectin and Visfatin/Nampt expression and MMP2, MMP9 and FSTL1 mRNA levels did not vary according to T2DM subgroups directly, leading to the assumption that these factors are not causal for scratch assay effects observed. In contrast significant mRNA expression differences were monitored between the thoracal tissue origins implying variations in the local effects of the different adipose tissue depots.

Effect of chlorhexidine skin disinfection and retrosternal gentamicin sponge on post-sternotomy mediastinitis: results from a prospective controlled registry of 2340 patients.

BACKGROUND: Reducing post-sternotomy mediastinitis (PSM) requires incorporating multiple methods. However, the independent effects of these measures are not well studied. AIM: To evaluate the independent effect of preoperative disinfection using isopropyl alcohol (IPA)-chlorhexidine gluconate (CHG) and the topical application of a retrosternal gentamicin collagen sponge at wound closure on reducing PSM. METHODS: From October 2012 to August 2014, 2340 patients were included in this prospective, controlled registry. Patients were divided into four groups. In groups 1 and 2, the skin was disinfected with IPA, and in groups 3 and 4, the skin was disinfected with IPA-CHG. A retrosternal gentamicin collagen sponge was used in groups 2 and 4. Freedom from PSM up to the 30th postoperative day was the primary endpoint. The secondary endpoint was freedom from any surgical site dehiscence. A stepwise regression model was made to reveal the independent factors associated with lower incidence of PSM. FINDINGS: There were significant differences in outcome among the groups (P < 0.0001). Primary healing was highest in group 4 (91.4%), which showed the lowest rate for mediastinitis (0.9%). Multivariate analysis showed that the use of CHG and a gentamicin sponge was statistically significant (P = 0.026 and 0.013, respectively). The other significant independent factors were valve operation (P = 0.001), body mass index >30 kg/m2 (P = 0.001), preoperative stroke (P = 0.005), and blood transfusion (P = 0.022). CONCLUSION: Preoperative skin disinfection with IPA-CHG is superior to only IPA, and it should be recommended. The addition of a retrosternal gentamicin-releasing sponge further reduces the rate of mediastinitis.

Camera-based photoplethysmography in critical care patients.

BACKGROUND: Camera-based photoplethysmography (cbPPG) is an optical measurement technique that reveals pulsatile blood flow in cutaneous microcirculation from a distance. cbPPG has been shown to reflect pivotal haemodynamic events like cardiac ejection in healthy subjects. In addition, it provides valuable insight into intrinsic microcirculatory regulation as it yields dynamic, two-dimensional perfusion maps. In this study, we evaluate the feasibility of a clinical cbPPG application in critical care patients. METHODS: A mobile camera set-up to record faces of patients at the bed site was constructed. Videos were made during the immediate recovery after cardiac surgery under standard critical care conditions and were processed offline. Major motion artefacts were detected using an optical flow technique and suitable facial regions were manually annotated. cbPPG signals were highpass filtered and Fourier spectra out of consecutive 10s signal segments calculated for heart rate detection. Signal-to-noise ratios (SNR) of the Fourier spectra were derived as a quality measure. Reference data of vital parameters were synchronously acquired from the bed site monitoring system. RESULTS: Seventy patient videos of an average time of 28.6±2.8 min were analysed. Heart rate (HR) was detected within a±5 bpm range compared to reference in 83% of total recording time. Low SNR and HR detection failure were mostly, but not exclusively, attributed to non-physiological events like patient motion, interventions or sudden changes of illumination. SNR was reduced by low arterial blood pressure, whereas no impact of other perioperative or disease-related parameters was identified. CONCLUSION: Cardiac ejection is detectable by cbPPG under pathophysiologic conditions of cardiovascular disease and perioperative medicine. cbPPG measurements can be seamlessly integrated into the clinical work flow of critical care patients.

Clinical course after surgical treatment of active isolated native mitral valve endocarditis.

AIM: Isolated mitral valve endocarditis (MVE) forms a particular subgroup within native infective valve endocarditis (NVE). We characterized this particular subgroup and analyzed the course of patients undergoing cardiac surgery. METHODS: Between 1997 and 2011, 474 patients underwent cardiac surgery at our institution for NVE treatment. Of these, 89 patients (18.8%) suffered from MVE. Valve replacement was undertaken in 84.2% and valve repair in 15.8%. Follow-up was completed with 267 patient years. RESULTS: A delay between the onset of first symptoms and surgery of 4.7±1.2 weeks was observed. Hence, most patients were in a critical preoperative state characterized by severe sepsis and destruction of the mitral valve. About 19.4% were emergency procedures. The MVE group presented with a higher prevalence of preoperative stroke, atrial fibrillation, coronary artery disease and chronic obstructive pulmonary disease in comparison with remaining NVE cases. MVE was more likely caused by Staphylococcus aureus; Staphylococcus epidermidis and Staphylococcus viridans were less frequent (P<0.01 each). Early mortality (6.7%) was caused by persistent sepsis. ICU stay >7 days and time on artificial ventilation >40 h led to a higher risk of in-hospital death. Five-year survival was 59.6% and affected by extracardiac comorbidities. CONCLUSION: Isolated MVE was characterized by a long delay before surgery, differences in microbiological findings and a higher prevalence of preoperative strokes in comparison to NVE. Surgery for MVE can be conducted with good clinical results, but mid-term outcome is limited by extracardiac comorbidities.

Prospective clinical trial on dosage optimizing of tranexamic acid in non-emergency cardiac surgery procedures.

UNLABELLED: After withdrawal of aprotinin in 2008 only tranexamic acid (TxA, Cyclocapron, Pfitzer, Germany) remains available as antihyperfibrinolytic agent in Europe. Dosage (from 1 g to 20 g) and application strategy (single shot i.v., infusion i.v., topical) reflect an indiscriminate use of TXA in cardiac surgery. We use data analysis of three registries to evaluate safety issues and sufficiency of different TxA dosages in our center. METHODS: Registry 1: Single shot ultra-low dose TxA (1 g in priming volume). Registry 2: Single shot medium dose TxA (5 g in priming volume). Registry 3: Single shot medium dose TxA (3 g in priming volume) and continuous, weight-adapted administration during cross clamping. Independence of surgeon's preference was achieved by changing dosage every surgery day regardless of operation schedule. RESULTS: Data analysis was carried out on 1182 consecutive, elective patients (1 g TxA n = 415; 3 g + x g TA n = 367; mean TxA dose 4.4 g ± 1.0 g; 5 g TxA n = 400). Patient characteristics were well matched in all three registries (mean age: 69 ± 9.5y, BMI 28.2 ± 4.7, Creatinin 107.5 ± 52.8 µM), as were performed surgical procedures (excluding organ transplantation). Postoperative data showed no significant differences for blood loss and major adverse events (1 g vs. 3 + g vs. 5 g: blood loss: 894 ± 1479 vs. 903 ± 1282 vs. 1004 ± 1604 ml; stroke: 1.5 vs. 1.6 vs. 1.5%; myocardial infarction 2.7 vs. 3.3 vs. 1.3%; 30d mortality 3.9 vs. 4.2 vs. 4.8%, respectively). Secondary endpoints (de novo dialysis, transfusion requirement, ICU and total treatment time) showed no significant differences between registries. CONCLUSION: Use of 1 g TxA is safe and sufficient for elective patients with on pump cardiac surgery and thus has been established as strategy of choice in our center.

[Surgery of the ascending aorta and aortic arch]. / Chirurgie der Aorta ascendens und des Aortenbogens.

The surgical therapy of the ascending aorta and the aortic arch with its supraarotic branches has been, and still is, one of the major challenges in the therapy of the aorta. There have been major developments in the endovascular therapy field, which can be used as a complementary or even sometimes as an alternative to the standard surgery. Therefore, an interdisciplinary approach is a prerequisite for optimal therapy planning for such patients. The risk of operating under circulatory arrest has been markedly reduced due to the continued development of cardiosurgical techniques, especially selective cerebral perfusion and mild hypothermia. Further developments of the therapy of aortic diseases via alternative approaches are expected in the near future. However, the conventional surgical therapy for pathologies in the ascending aorta (with/out the aortic valve) and the aortic arch is still the gold standard.

Influence of systemic hypothermia on the myocardial oxygen tension during extracorporeal circulation: comparative study in German Landrace pigs.

During extracorporeal circulation (ECC) controlled hypothermia is a common method of myocardial protection due to a reduction of the myocardial oxygen consumption. Although the beneficial aspects of hypothermia on the myocardial metabolism have been widely demonstrated the effect of hypothermia on the myocardial oxygen tension (PmyO2) is unclear. For this reason the PmyO2 of German Landrace pigs (male, three months of age) during ECC was analysed under mild hypothermia (32°C, n = 6 pigs) and under normothermia (n = 10 pigs, control group) within a time period of 23 min (1400 sec). Flexible invasive Clark type microcatheters were used to measure the PmyO2 in the beating heart. During normothermal ECC a continuous PmyO2 increase from 36.5 ± 15.8 mmHg to 52.6 ± 27.2 mmHg (+44.1%) after 1400 sec was measured (p = 0.02). In contrast, mild hypothermia caused a continuous PmyO2 decrease from initially 46.9 ± 17.5 mmHg to 36.7 ± 20.8 mmHg (-21.8%, p < 0.013) in the test period. Electrocardiography revealed no signs of ischemia or arrhythmia during normo- and hypothermic ECC. It seems obvious that mild hypothermia results in a reduction of the oxygen transfer to the myocardial cells and that this effect outweighs the beneficial effects of hypothermia in the myocardium which are related to reduced oxygen consumption. However, in mild hypothermia oxygen supply to the myocardium remained sufficient for normal myocardial function.

Cutaneous and muscular microcirculation in patients with terminal heart failure awaiting transplantation.

Heart failure patients are clinically characterized by extreme cardiomegaly, breathlessness, fluid retention and an early onset of fatigue. Studies have shown generalized restricted blood flow in those patients. Furthermore animal experiments proved an impaired blood flow and a diminished oxygen supply of the skeletal muscle in animals with chronic heart failure. Patients with chronic heart failure are limited to the extent of their ability to regulate their arterial pressure, especially in physical activity. It is however unclear in what way restriction of blood flow in the main arteries correlates with those in capillaries and to what extent. In this study it was examined the depth of capillary circulatory restriction as well as the disregulation of oxygen partial pressure in skeletal muscle in rest and stress conditions, in patients with terminal heart failure.

Skin sealant InteguSeal® has no impact on prevention of postoperative mediastinitis after cardiac surgery.

BACKGROUND: Surgical site infections (SSIs) after median sternotomy represent a serious complication and a high potential risk for adverse clinical outcome after cardiac surgery. The antimicrobial skin sealant InteguSeal® was introduced as a novel tool in preventing development of SSI. AIM: This single-centre investigation used two prospective registries to evaluate the prophylactic effect of a cyanoacrylate-based antimicrobial skin sealant (InteguSeal®) on the incidence of postoperative mediastinitis or any other form of chest skin incision SSI after elective cardiac surgery. METHODS: Between October 2010 and April 2011 a total of 998 patients underwent elective cardiac surgical procedures with median sternotomy in our centre. In 496 patients InteguSeal® was included in standard preoperative preparation procedures before chest skin incision (group 1). In 502 patients standard preoperative skin preparation procedures were used without InteguSeal® (group 2). Freedom from mediastinitis and from any other form of SSI within 30 postoperative days were the primary and secondary endpoints, respectively. FINDINGS: A total of 983 patients were eligible for inclusion in per-protocol analysis (488 vs 495 patients). The incidence of postoperative mediastinitis was 2.3% in group 1 vs 3.2% in group 2 (not significant). The incidence of any form of SSI was 10.9% in group 1 vs 11.5% in group 2 (not significant). Perioperative patient characteristics, complexity of surgical procedures performed and length of hospitalization were similar in both groups. CONCLUSION: The use of InteguSeal® has no influence on the incidence of postoperative SSI and mediastinitis after cardiac surgery with median sternotomy.

Readmission to the intensive care unit after cardiac surgery: a single-center experience with 7105 patients.

AIM: Identification of patients at high risk for readmission to the Intensive Care Unit (ICU) after cardiac surgery is paramount. We evaluated the clinical characteristics of readmitted patients and identified perioperative prognostic variables for ICU readmission. METHODS: A total of 7105 patients who underwent cardiac surgery between 2007 and 2010 and discharged after a primary stay in the ICU were reviewed retrospectively. Of these, 7.8% (554) patients were readmitted. The reasons for readmission and postoperative course were analyzed. Perioperative risk factors for readmission were determined by multivariate regression analysis. RESULTS: Mortality of patients after readmission was 13.6% compared with 0.2% without recidivism (P<0.0001). Mean length of stay in hospital of patients requiring readmission was 24.9 ± 19.1 days and significantly longer compared to all other patients 12.3±8.4 days (P<0.0001). The main reasons for readmission were respiratory failure (39.0%) and cardiovascular instability (26.2%). Complex cardiac surgery, aortic surgery and extended stay in the ICU were the most powerful variables to predict ICU readmission. CONCLUSION: ICU readmission was related to complex surgery and associated with impaired outcome. Respiratory complications were the most common reasons for readmission. Predictive renal and pulmonary risk factors indicate the need of preoperative preconditioning and patient selection.

The C-Port xV® vascular anastomosis system: results from an animal trial.

BACKGROUND: In this study, facilitated anastomosis using an anastomotic device was compared to conventional hand-sewn (HS) vascular anastomosis in an animal model. METHODS: A pig carotid bypass model was employed. C-Port xV® (xV) and HS anastomoses were compared by evaluating intraoperative performance, midterm graft patency, and histology. RESULTS: All animals survived; none developed early/late neurological deficits. Mean graft blood flow was comparable between groups (HS group: 161 ± 61 ml/min; xV group: 143 ± 44 ml/min). All anastomoses were patent at necropsy (at 111 ± 6 postoperative days). Histologically, no significant inflammation was found around the fasteners or in the vessel wall. Neointimal overgrowth on the lumen surface appeared organized and covered with endothelium. There was no adherence of fibrin, platelets, or inflammatory cells to the surface. The neointimal tissue appeared normal without any inflammation, hemorrhage, calcification, or necrosis. CONCLUSION: Facilitated vascular anastomosis using the xV anastomotic device is safe and effective in the pig carotid bypass model. Further studies should evaluate the efficacy of this device when used in confined spaces to define its potential role in minimally invasive procedures.

Intra-aortic balloon pump implantation does not affect long-term survival after isolated CABG in patients with acute myocardial infarction.

BACKGROUND: Intra-aortic balloon pump (IABP) is an established therapy to support patients with heart failure during coronary artery bypass grafting (CABG). The impact of the timing of IABP on the hospital course and on follow-up is of particular clinical interest. The purpose of this study was to analyze the relationship between the time of IABP implantation and its impact on early, mid- and long-term survival in patients with acute myocardial infarction (AMI) who underwent emergent CABG for NSTEMI and STEMI. METHODS: A total of 472 patients with AMI (NSTEMI and STEMI) underwent emergency CABG at our institution; 158 of them additionally received IABP support. Fifty-seven (36 %) patients received preoperative and 101 (64 %) patients underwent intraoperative IABP implantation. Endpoints were in-hospital und follow-up (mean duration 37 ± 28 months) survival. RESULTS: Overall in-hospital mortality was 17.1 % (n = 27): 17.6 % (n = 10) in the preoperative group and 16.8 % (n = 17) in the intraoperative group ( P = ns). Mid- and long-term survival rates were comparable for both groups 78.6 % vs. 73.7 %, 71.4 % vs. 68.7 % and 64.3 % vs. 54.6 % at 1, 3 and 5 years, respectively ( P = ns). CONCLUSION: This study demonstrates that CABG with IABP support in high-risk patients with AMI can be performed with acceptable in-hospital and long-term survival rates. The decision for IABP placement should consider the preoperative clinical condition and the intraoperative course of each patient. IABP placement does not appear to affect the long-term outcome after isolated CABG in patients with AMI.

[Transapical aortic valve implantation--indications, risks and limitations]. / Transapikaler Aortenklappenersatz--Indikationen, Risiken und Grenzen.

Calcified aortic stenosis is the predominant valve disease in the western world. Currently, surgical aortic valve replacement is the gold standard procedure for symptomatic severe aortic stenosis that can be performed with low morbidity and mortality. The prevalence of aortic stenosis increases with age, and the incidence of several comorbidities also unavoidably elevates the risk of surgical treatment. Therefore, the most adequate and gentle treatment is needed especially for this population. Since the first transcatheter aortic valve implantation (TAVI) was performed in 2002, the main implanting routes are the transfemoral, retrograde access through the common femoral artery, and the antegrade, transapical approach via anterolateral minithoracotomy. Meanwhile, TAVI has become an alternative treatment for patients who are not suitable candidates for surgical therapy in some centers.The initial clinical results are promising and have confirmed the feasibility of this technique. Due to the restricted long-term data, conventional aortic valve replacement still remains the standard for the treatment of aortic stenosis. Selection of the suitable therapy approach (surgical replacement, transfemoral or transapical aortic valve implantation) must consider each patient's specific risk profile and individual indication. Prospective, randomized trials will be necessary to assess the individual survival benefit of TAVI for various risk populations and to extend the indication.

Critical hematocrit and oxygen partial pressure in the beating heart of pigs.

In cardiac surgery the substitution of lost blood volume by plasma substitutes is a common therapeutical approach. None of the currently available blood substitutes has a sufficient oxygen transport capacity. This can limit the functional integrity of the myocardium known as highly oxygen consumptive. The study was aimed to get information about the minimal hematocrit, also known as critical hematocrit (cHct), which guarantees a stable and adequate oxygen partial pressure in the myocardium (pO2). In adult female pigs (n=7) the hematocrit was reduced by isovolemic blood dilution with an intravenous infusion of isotonic 4% gelatine polysuccinate solution, The substituted blood volume ranged between 3000ml and 7780ml (mean: 5254±1672ml). In all animals the pO2 of the myocardium of the beating heart and of the resting skeletal muscle increased until blood dilution resulted in a Hct decrease down to 15%. Further blood dilution resulted in a decrease of the pO2. Only after the Hct was <10% the pO2 was lower than before blood dilution and accompanied by a lethal ischemia of the myocardium. These data indicate a cHct of about 10% in the pig animal model.

Immunosuppression using the mammalian target of rapamycin (mTOR) inhibitor everolimus: pilot study shows significant cognitive and affective improvement.

UNLABELLED: Immunosuppression using calcineurin inhibitors (CNIs) is accompanied by neuropsychiatric side effects, which counteract longevity and quality of life benefits in 10% to 28% of patients. Following the availability of the mammalian target of rapamycin (mTOR) inhibitors, it became possible to replace CNI without increasing the risk of acute graft rejection. mTOR, a member of the phosphatidyl inositol 3' kinase family, is a downstream target of brain-derived neurotrophic factor, which has been implicated in the pathophysiology and treatment of several psychiatric disorders. Preclinical evidence has implicated the mTOR pathway in synaptic plasticity and fear memory consolidation and reconsolidation. METHODS: In the present study we prospectively evaluated the psychiatric outcomes of CNI-free immunosuppression in adult maintenance heart transplant recipients (n = 9; age: 66.1 +/- 6.1) using the Wechsler Memory Scale-Revised (WMS-R), Symptom Checklist-90-Revised (SCL-90-R), Beck Depression Inventory (BDI), Trail Making Tests A and B, Digit Span (DS), and Hamilton Depression Scale (HAMD). RESULTS: Four weeks after switching to CNI-free immunosuppression using everolimus, BDI (Z = -1.14; P = .048), Trail Making tests A and B (Z = -2.52; P = .012), WMS-R (Z = 2.37; P = .018), and SCL-90-R (Z = -2.37; P = .018) were all significantly improved while DS (Z = -1.18; P = .236) and HAMD (Z = -0.595; P = .552) remained unchanged. CONCLUSION: This report describes favorable psychiatric outcome variables using everolimus in maintenance heart transplant recipients. CNI-free immunosuppression with everolimus might provide significant improvement in memory, concentration, and overall psychiatric symptoms among heart transplant recipients.