Intravascular Application of Labelled Cell Spheroids: An Approach for Ischemic Peripheral Artery Disease.

Mesenchymal stem cells (MSC) are known for their vascular regeneration capacity by neoangiogenesis. Even though, several delivery approaches exist, particularly in the case of intravascular delivery, only limited number of cells reach the targeted tissue and are not able to remain on site. Applicated cells exhibit poor survival accompanied with a loss of functionality. Moreover, cell application techniques lead to cell death and impede the overall MSC function and survival. 3D cell spheroids mimic the physiological microenvironment, thus, overcoming these limitations. Therefore, in this study we aimed to evaluate and assess the feasibility of 3D MSCs spheroids for endovascular application, for treatment of ischemic peripheral vascular pathologies. Multicellular 3D MSC spheroids were generated at different cell seeding densities, labelled with ultra-small particles of iron oxide (USPIO) and investigated in vitro in terms of morphology, size distribution, mechanical stability as well as ex vivo with magnetic resonance imaging (MRI) to assess their trackability and distribution. Generated 3D spheroids were stable, viable, maintained stem cell phenotype and were easily trackable and visualized via MRI. MSC 3D spheroids are suitable candidates for endovascular delivery approaches in the context of ischemic peripheral vascular pathologies.

Molecular classification of primary mediastinal large B-cell lymphoma using routinely available tissue specimens.

Primary mediastinal large B-cell lymphoma (PMBCL) is recognized as a distinct entity in the World Health Organization classification. Currently, the diagnosis relies on consensus of histopathology, clinical variables, and presentation, giving rise to diagnostic inaccuracy in routine practice. Previous studies have demonstrated that PMBCL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures. However, requirement of fresh-frozen biopsy material has precluded the transfer of gene expression-based assays to the clinic. Here, we developed a robust and accurate molecular classification assay (Lymph3Cx) for the distinction of PMBCL from DLBCL subtypes based on gene expression measurements in formalin-fixed, paraffin-embedded tissue. A probabilistic model accounting for classification error, comprising 58 gene features, was trained on 68 cases of PMBCL and DLBCL. Performance of the model was subsequently evaluated in an independent validation cohort of 158 cases and showed high agreement of the Lymph3Cx molecular classification with the clinicopathological diagnosis of an expert panel (frank misclassification rate, 3.8%). Furthermore, we demonstrate reproducibility of the assay with 100% concordance of subtype assignments at 2 independent laboratories. Future studies will determine Lymph3Cx's utility for routine diagnostic purposes and therapeutic decision making.

Pull-off characteristics of double-shanked compared to single-shanked ligation clips: an animal study.

BACKGROUND: The use of surgical ligation clips is considered as the gold standard for the closure of vessels, particularly in laparoscopic surgery. The safety of clips is mainly achieved by the deep indentation of the metal bar with a high retention force. A novel double-shanked (DS) titanium clip was compared to two single-shanked clips with respect to axial and radial pull-off forces. METHODS: In a porcine model (8 animals, 51±1 kg), arteries were prepared immediately after euthanisation, assigned to either a medium (2-4 mm; n=120) or a medium-large (3.5-7 mm; n=120) clip size group, and clipped with the appropriate clip size. After dissection, axial and radial pull-off forces were measured. RESULTS: The axial pull-off force of the DS-Clip was higher than one single-shanked clip and comparable to the other single-shanked clip, and overall was linearly correlated to the cross-sectional area of the clip. The radial pull-off force of the DS-Clip was significantly higher than both single-shanked clips and, for the single-shanked clips, was correlated to the total clip thickness. The variation of radial pull-off force was lower for the DS-Clip due to a defined catch in the clip applier. CONCLUSIONS: The radial pull-off force was lower than the axial pull-off force in total and therefore appears to be the critical point of dislocation. Due to the higher total holding mass, the DS-Clip provided a clear advantage in this regard and might therefore decrease the dislocation rate. The catch in the applier increases the reproducibility in clip placement.

First In Vivo Results of a Novel Pediatric Oxygenator with an Integrated Pulsatile Pump.

Extracorporeal membrane oxygenation (ECMO) is a pivotal bridge to recovery for cardiopulmonary failure in children. Besides its life-saving quality, it is often associated with severe system-related complications, such as hemolysis, inflammation, and thromboembolism. Novel oxygenator and pump systems may reduce such ECMO-related complications. The ExMeTrA oxygenator is a newly designed pediatric oxygenator with an integrated pulsatile pump minimizing the priming volume and reducing the surface area of blood contact. The aim of our study was to investigate the feasibility and safety of this new ExMeTrA (expansion mediated transport and accumulation) oxygenator in an animal model. During 6 h of extracorporeal circulation (ECC) in pigs, parameters of the hemostatic system including coagulation, platelets and complement activation, and flow rates were investigated. A nonsignificant trend in C3 consumption, thrombin-antithrombin-III (TAT) complex formation and a slight trend in hemolysis were detected. During the ECC, the blood flow was constantly at 500 ml/min using only flexible silicone tubes inside the oxygenator as pulsatile pump. Our data clearly indicate that the hemostatic markers were only slightly influenced by the ExMeTrA oxygenator. Additionally, the oxygenator showed a constant quality of blood flow. Therefore, this novel pediatric oxygenator shows the potential to be used in pediatric and neonatal support with ECMO.

Sympathetic activity-associated periodic repolarization dynamics predict mortality following myocardial infarction.

BACKGROUND: Enhanced sympathetic activity at the ventricular myocardium can destabilize repolarization, increasing the risk of death. Sympathetic activity is known to cluster in low-frequency bursts; therefore, we hypothesized that sympathetic activity induces periodic low-frequency changes of repolarization. We developed a technique to assess the sympathetic effect on repolarization and identified periodic components in the low-frequency spectral range (≤0.1 Hz), which we termed periodic repolarization dynamics (PRD). METHODS: We investigated the physiological properties of PRD in multiple experimental studies, including a swine model of steady-state ventilation (n=7) and human studies involving fixed atrial pacing (n=10), passive head-up tilt testing (n=11), low-intensity exercise testing (n=11), and beta blockade (n=10). We tested the prognostic power of PRD in 908 survivors of acute myocardial infarction (MI). Finally, we tested the predictive values of PRD and T-wave alternans (TWA) in 2,965 patients undergoing clinically indicated exercise testing. RESULTS: PRD was not related to underlying respiratory activity (P<0.001) or heart-rate variability (P=0.002). Furthermore, PRD was enhanced by activation of the sympathetic nervous system, and pharmacological blockade of sympathetic nervous system activity suppressed PRD (P≤0.005 for both). Increased PRD was the strongest single risk predictor of 5-year total mortality (hazard ratio 4.75, 95% CI 2.94-7.66; P<0.001) after acute MI. In patients undergoing exercise testing, the predictive value of PRD was strong and complementary to that of TWA. CONCLUSION: We have described and identified low-frequency rhythmic modulations of repolarization that are associated with sympathetic activity. Increased PRD can be used as a predictor of mortality in survivors of acute MI and patients undergoing exercise testing. TRIAL REGISTRATION: ClinicalTrials.gov NCT00196274. FUNDING: This study was funded by Angewandte Klinische Forschung, University of Tübingen (252-1-0).

Internally gas-cooled radiofrequency applicators as an alternative to conventional radiofrequency and microwave ablation devices: an in vivo comparison.

PURPOSE: To test the efficacy of internally CO2-cooled radiofrequency (RF) ablation in vivo and to compare its effectiveness to a standard water-cooled RF probe and to a gas-cooled microwave (MW) device. METHOD AND MATERIALS: 49 ablations were performed on 15 pigs under general anesthesia using 15G monopolar CO2-cooled RF applicators, 17G monopolar water-cooled RF applicators and 15G internally CO2-cooled microwave devices. The power of the MW device was 45W, the current of the gas-cooled RF device was 1200-1600mA. At the water-cooled RF probe, maximum power of 200W was set. Ablation time was 15min. The short and long axes of the ablation zone were measured. Histological analyses and NADH-staining were performed. The diameters and the ablation volumes were compared using an analysis of variance. RESULTS: No spots of untreated tissue were observed close to the cooled needle track in any of the ablation zones. The largest short axis diameter was 3.4±0.5cm achieved with the gas-cooled monopolar applicator. With the water-cooled applicators, short axis diameter was significantly smaller, reaching 2.5±0.4cm. Gas-cooled MW probes achieved 2.9±1.0cm. The largest ablation volume was 31.5±12ml (gas-cooled RF), and the smallest was 12.7±4ml (water-cooled RF). Short/long axis ratio was largest for gas-cooled RF probes with 0.73±0.08 versus 0.64±0.04 for the water-cooled probes and 0.49±0.25 for the microwave applicator. CONCLUSION: Gas-cooled RF applicators may have a higher potential for effective destruction of liver lesions than comparable water-cooled RF systems, and may be an alternative to standard RF and MW ablation devices.

Balloon coating with rapamycin using an on-site coating device.

PURPOSE: The efficacy of drug-eluting balloons has been demonstrated in clinical trials. The drug predominantly used is paclitaxel because of its lipophilic properties and the rapid onset of action. The aim of the investigation was to evaluate the feasibility and efficacy of an alternative balloon coating with rapamycin that can be applied on site. METHODS: The balloon coating (3.0/18 and 3.0/12 mm, Cathy No. 4, Translumina GmbH) with rapamycin was conducted with a coating machine (Translumina GmbH). Concentrations were 2, 2 × 2, 3, and 4 %. Measurements regarding the amount of substance released to the vessel wall were carried out on explanted porcine coronaries by means of ultraviolet and visible-light spectroscopy. Inflation time varied between 30 and 120 s. The biological effect of the coating was evaluated in a porcine peripheral overstretch and stent implantation model. RESULTS: The amount of rapamycin on the balloon surface ranged from 558 ± 108 µg for the 2 % solution to 1,441 ± 228 µg in the 4 % solution. An amount of 95 ± 63-193 ± 113 µg was released into the vessel wall. The quantitative measurements of the angiographic examinations 4 weeks after treatment revealed a reduction of diameter stenosis from 20.6 ± 17.4 % in the control group to 11.6 ± 5.5 % in the drug-eluting balloon group. CONCLUSION: A balloon coating with rapamycin omitting an excipient is possible with a dose-adjustable coating machine. However, the biological effects are moderate, which make further optimization of the coating process and evaluation of appropriate excipients necessary.

Genomic aberrations affecting the outcome of immunodeficiency-related diffuse large B-cell lymphoma.

The objective of this study was to evaluate the prognostic impact of genomic regions in a series of human immunodeficiency virus (HIV)-related diffuse large B-cell lymphomas (HIV-DLBCLs) and post-transplant DLBCLs (PT-DLBCLs) analyzed by genome-wide DNA profiling. Minimal common regions (MCRs) were estimated on genomic profiles obtained using Affymetrix Human Mapping 250k Nsp I arrays and tested for their impact on clinical outcome by univariate analysis on 36 PT-DLBCLs, 19 HIV-DLBCLs and, as a control group, 149 DLBCLs arising in immunocompetent individuals (IC-DLBCLs). PT-DLBCL and HIV-DLBCL presented a similar outcome. Immunodeficiency-related DLBCL (ID-DLBCL) had a worse overall survival (OS) than IC-DLBCL. Seven MCRs showed a statistical impact on OS in PT-DLBCL and four in HIV-DLBCL. Among these, the presence of gains at 1q or at 18q defined a group of patients with PT-DLBCL with a very poor outcome (p < 0.0001). The presence of del(3p14.2) or of + 2p23.1 identified a group of HIV-DLBCLs with a very poor outcome (p = 0.0072). It was concluded that genomic aberrations affecting outcome differ between ID-DLBCL and IC-DLBCL and are also dependent on the type of acquired immunodeficiency.

Assessing acute ventricular volume changes by intracardiac impedance in a chronic heart failure animal model.

BACKGROUND: Implantable device diagnostics may play an essential role in simplifying the care of heart failure patients by providing fundamental insights into their complex clinical patterns. Early recognition of heart failure progression by a continuous hemodynamic monitoring would allow for timely therapeutic interventions to prevent decompensation and hospitalization. In this study, the feasibility of assessing ventricular volume changes by implant-based measurements of intracardiac impedance was tested in a heart failure animal model. METHODS: Heart failure was induced in five minipigs by high-rate pacing over 3 weeks. During a final open-chest examination a graded dobutamine stress test was performed. Stroke volume (SV) was measured by an ultrasonic flow probe at the ascending aorta. End diastolic pressure (EDP) and maximum pressure slope (dP/dtmax) were calculated from a left ventricular microtip catheter signal. Impedance was measured by an implanted pacemaker between biventricular leads. Stroke impedance (SZ) was calculated as the difference between end-systolic and end-diastolic impedance (EDZ). RESULTS: Administration of dobutamine led to an increase in SV (55+/-16%), dP/dtmax (107+/-89%), and SZ (56+/-30%). EDP changed by 37+/-21% whereas EDZ changed by 7.4+/-4%. Significant correlations were found between SZ and SV (r=0.88), and between EDZ and EDP (r=-0.82). CONCLUSION: The strong correlation with SV allows the application of intracardiac impedance measurements for an implant-based continuous monitoring of cardiac function. Impedance may also be used for hemodynamic optimization of cardiac resynchronization therapy.

Immobilized DNA aptamers used as potent attractors for porcine endothelial precursor cells.

Because of their insufficient biocompatibility and high thrombogenicity, small diameter artificial vascular prostheses still do not show a satisfactory patency rate. In vitro endothelialization of artificial grafts before implantation has been established experimentally years ago, but, this procedure is extremely time consuming and expensive. This study deals with the coating of graft surfaces with capture molecules (aptamers) for circulating endothelial progenitor cells (EPCs), mimicking a prohoming substrate to fish out EPCs from the bloodstream after implantation and to create an autologous functional endothelium. Using the SELEX technology, aptamers with a high affinity to EPCs were identified, isolated, and grafted onto polymeric discs using a blood compatible star-PEG coating. A porcine in vitro model that demonstrates the specific adhesion of EPCs and their differentiation into vital endothelial-like cells within 10 days in cell culture is presented. We suggest that the rapid adhesion of EPCs to aptamer-coated implants could be useful to promote endothelial wound healing and to prevent increased neointimal hyperplasia. We hypothesize that future in vivo self-endothelialization of blood contacting implants by homing factor mimetic capture molecules for EPCs may bring revolutionary new perspectives towards clinical applications of stem cell and tissue engineering strategies.

Perfusionists in the world of tissue engineering--a quick and easy method for safe isolation of huge quantities of porcine cardiomyocytes.

Isolation of huge quantities of primary cells from whole organs like the heart becomes increasingly important, especially for the emerging research field of tissue engineering and regenerative medicine. This study deals with the isolation of pig cardiomyocytes, in contrast to the standard mouse or rat models, because we aimed to draw attention to the species, which are genetically more closely related to the human organism. The bigger operative and veterinary expenditure of the pig-heart model can only be justified by a technique that supplies a big amount of qualitative high-grade cardiomyocytes. In our model, the quality is guaranteed by protection of the heart, already in situ, by a cardioplegia and a careful application of collagenase to soften the tissue. The construction of a new apparatus which includes enormous costs was not necessary, since the perfusion equipment was realized from two commercially available HLMsets, which were modified and connected to each other. Our model makes it possible to rinse the whole myocardium, which leads to a better output than models that only prepare a part of the myocardium around a coronary artery. The careful harvesting of high-grade cardiomyocytes is an important source of successful cell cultures to be used for numerous experimental applications, may reduce animal experiments and, additionally, represents a chance for perfusionists to become an important partner in interdisciplinary research projects.

Intracardiac impedance monitors hemodynamic deterioration in a chronic heart failure pig model.

INTRODUCTION: A large number of heart failure (HF) patients benefit from cardiac resynchronization therapy. Measurements of intrathoracic impedance (ITZ) by implantable devices correlate with intrathoracic fluid content and are used for monitoring lung edema formation in HF patients. However, intrathoracic fluid is only an indirect parameter of cardiac function. We hypothesized that changes in intracardiac impedance correlate with left ventricular (LV) volume changes. Therefore, measurements of intracardiac impedance between a right ventricular lead and a LV lead may be used to monitor long-term changes of LV function. METHODS AND RESULTS: HF was successfully induced in nine mini-pigs by continuous high-rate pacing. Hemodynamic parameters as well as intracardiac impedance and ITZ were measured before HF induction and after 20 +/- 5 days of high-rate pacing. After the pacing period, we found a significant deterioration of hemodynamics, reflected by a reduction of ejection fraction from 71+/-11% to 48+/-7% and an increase of LV end diastolic pressure (EDP) from 12 +/- 4 mmHg to 26 +/- 8 mmHg. Worsening of cardiac function correlated with a significant >30% decrease of end diastolic intracardiac impedance, in accordance with a >20% increase of end diastolic volume (EDV). ITZ decreased by more than 8%. We observed a significant inverse correlation between end diastolic intracardiac impedance and EDP (r =-0.81, P < 0.001). CONCLUSIONS: In this animal model, changes of intracardiac impedance revealed hemodynamic deterioration as reflected by EDV and EDP pressure. Thus, intracardiac impedance is a promising new application to monitor heart failure status within implantable devices.

Compound subretinal prostheses with extra-ocular parts designed for human trials: successful long-term implantation in pigs.

BACKGROUND: Subretinal implants aim to replace photoreceptor function in patients suffering from degenerative retinal disease like retinitis pigmentosa by topically applying electrical stimuli in the subretinal space. This study-as a last step before upcoming human trials-explored a newly developed surgical technique for permanent implantation of complex subretinal implants with extra-ocular parts. METHODS: The implant consisted of a microphoto-diode array (MPDA) with 1550 electrodes and a 4x4 array of gold electrodes for direct electrical stimulation; both were mounted onto a polyimide foil for transscleral placement into the subretinal space. The foil carried connection lanes to a silicone cable that was implanted under the skin and led to a stimulator box in the animal's neck. Surgery was performed in 11 domestic pigs. Improved vitreo-retinal surgical technique consisted of a 180 degrees peripheral retinotomy and use of diathermy to penetrate the choroid in order to avoid choroidal haemorrhage. Subretinal forceps were used to place the implant safely onto the retinal pigment epithelium before the retina was flattened, peripheral laser photocoagulation was applied and the eye was filled with silicon oil. The implant was stabilized by a scleral fixation patch, use of a metal clamp with bone screws on the animal's skull and a tissue ring under the animal's skin in the neck. Behaviour was observed in the freely moving animals after direct subretinal electrical stimulation and funduscopy, optical coherence tomography, fluorescein angiography and histology were performed. RESULTS: All implants were successfully placed subretinally. In three animals a proliferative vitreo-retinopathy was observed after approximately 2 weeks. Otherwise, funduscopy and OCT demonstrated complete retinal attachment and FA showed no retinal vascular abnormalities over and around the implant. The animals showed clear behavioural reactions to electrical stimulation over the whole examination period. Histological examination failed to show any voltage-induced alteration in the cellular architecture of the retina overlying the stimulation electrodes. CONCLUSIONS: This study demonstrates the feasibility of a new surgical procedure for highly safe and controlled implantation of complex subretinal devices with extra-ocular parts. The new implant design proved to be safely implantable in free-moving pigs for an observation period of 4 weeks.

Resection of the inferior superior turbinate: does it affect olfactory ability or contain olfactory neuronal tissue?

BACKGROUND: One approach to the sphenoid sinus involves resection of the inferior portion of the superior turbinate. There is general agreement from anatomic investigations that this area contains olfactory mucosa. This study will determine if olfactory tissue can be found in the superior turbinate mucosa of patients with chronic sphenoiditis and what effect its removal has on a patient's olfactory ability. METHODS: The inferior one-third of the superior turbinate removed during endoscopic sphenoidotomy was stained with olfactory marker protein antibody, a marker for mature olfactory tissue. The specimens were graded for content of olfactory neuronal elements. All patients underwent uninasal 12-item smell identification testing before surgery and at least 3 weeks after surgery. RESULTS: Fifty-five superior turbinate samples were taken from 31 patients. Nine (16%) of 55 samples contained olfactory neuronal elements that stained with olfactory marker protein. When comparing the pre- and postoperative smell test results, 52% of the nostrils had no more than a one-item change, 35% of the nostrils had a more than one-item improvement, and only 12% had more than a one-item loss. None of the nostrils with a loss of olfactory ability after the surgery showed olfactory neuronal elements in their superior turbinate specimens. CONCLUSION: There is olfactory mucosa in approximately one-sixth of the superior turbinate specimens removed during the endoscopic transethmoidal sphenoidotomy procedure. Although 12% of the patients had a loss of olfactory ability in this study, none of the loss could be attributed to excision of olfactory tissue.