Ex-vivo Conditions Research Articles

Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth

Patterning of neuronal outgrowth in vitro is important in tissue engineering as well as for the development of neuronal interfaces with desirable characteristics. To date, this has been achieved with the aid of micro- and nanofabrication techniques giving rise to various anisotropic topographies, either in the form of continuous or discontinuous structures. In this study we propose a currently unexplored geometry of a 3D culture substrate for neuronal cell growth comprising discontinuous subcellular microstructures with anisotropic geometrical cross-section. Specifically, using laser precision 3D micro/nano fabrication techniques, silicon substrates comprising arrays of parallel oriented elliptical microcones (MCs) were fabricated to investigate whether a discontinuous geometry comprising anisotropic features at the subcellular level could influence the alignment of peripheral nervous system cell populations. It was shown that both Schwann cells and axons of sympathetic neurons were parallel oriented onto the MCs of elliptical shape, while they exhibited a random orientation onto the MCs of arbitrary shape. Notably, this topography-induced guidance effect was also observed in more complex cell culture systems, such as the organotypic culture whole dorsal root ganglia (DRG) explants. Our results suggest that a discontinuous topographical pattern could promote Schwann cell and axonal alignment, provided that it hosts anisotropic geometrical features, even though the sizes of those range at the subcellular lengthscale. The laser-patterned arrays of MCs presented here could potentially be a useful platform for patterning neurons into artificial networks, allowing the study of neuronal cells interactions under 3D ex-vivo conditions.

A reference dataset of in-vivo human left-ventricular fiber architecture in systole and diastole

Background Computational cardiac modelling has been established as a valuable tool for simulating electrophysiology and electromechanics of the heart [1], with promising applications to “personalized medicine” [1]. Realistic computational models require a detailed description of left-ventricular cardiac fiber architecture. So far, nonpatient-specific fiber architectures have been obtained from histology or diffusion tensor imaging (DTI) of excised post-mortem hearts. However, compared to invivo, ex-vivo physiological conditions including ventricular pressure and residual contractile forces deviate significantly, hence potentially impacting measured fiber metrics. The objective of this work was to obtain and make available cardiac DTI data of the in-vivo human heart with full cardiac coverage in both peak systole and mid diastole including correction for myocardial strain.

LspA gene of Mycobacterium tuberculosis co-transcribes with Rv1540 and induced by surface and acidic stress

Lipoprotein signal peptidase, lspA (Rv1539), is the only known gene in mycobacterial genome for cleaving the signal sequence from prolipoprotein to form mature lipoprotein. It has been implicated in maintaining the virulence of Mycobacterium tuberculosis. The regulation of lspA had not been studied so far. Here, we identify a novel operon lspA–Rv1540 in M. tuberculosis. We detected co-transcription of the open reading frames of lspA–Rv1540 in in-vitro as well as in ex-vivo conditions. Analysis of the sequence upstream to lspA revealed a strong promoter activity that was shown to be induced significantly by surface stress and acidic environment.

Role of Synovial Fluid Constituents on Tribological Behavior of Synovial Joints - Application for Developing Novel Therapeutic Substitutes

Joint diseases represent a major health problem because they evolve towards the wear of cartilage for which no treatment is really effective. The difficulties in identifying the causes of these diseases are related to the biochemical and structural complexity of synovial fluid that allow cartilage lubrication. Thereby, recent studies show that synovial fluid contains micro-vesicles filled with a glycoprotein gel (hyaluronic acid and protein) surrounded by stacks of lipid bilayers which gives it excellent lubricating properties [1]. On the other side, this stable structure in vivo, becomes unstable in the ex vivo conditions. In this context, the study aims to tests the lubricating properties of synovial fluid constituents in order to identify their role on the tribological behavior of synovial joints and to develop a biomimetic synovial fluid using self-assembly of polyelectrolytes in order to stabilize the ex vivo structure for the potential therapeutic synovial fluid substitutes.Our results show that the new synthetized polysaccharides influence the friction coefficient, the stability and the wear of lipids bilayers, providing lubricating properties superior to synovial fluid biological constituents. In addition, their capacity to be structured in micro-spheres and included in lipid micro-vesicles indicates them as good biomimetic lubricants. This will allow a longer stability of synovial fluid substitutes in ex-vivo conditions in order to achieve the best lubricating properties and to improve the joint diseases treatment.

A major phospholipase A2 from Daboia russelii russelii venom shows potent anticoagulant action via thrombin inhibition and binding with plasma phospholipids

This is the first report on antithrombin effects of a phospholipase A2 (RVAPLA2) purified from venom of Daboia russelii russelii. The N-terminal sequence as well as in-gel tryptic digested peptides of RVAPLA2 showed significant homology with PLA2s from Russell's viper venom. RVAPLA2 demonstrated highest specific activity in hydrolyzing phosphatidylcholine (1.8×10(6)U/mg) with Km and Vmax values of 0.61mM and 132.3μmol/min, respectively. RVAPLA2 exerted dose-dependent catalytic and strong anticoagulant activities; however, studies indicated dissociation of its catalytic and anticoagulant sites. The anticoagulant action of RVAPLA2 was partially contributed by catalytic hydrolysis of plasma phospholipids. RVAPLA2 showed strong anticoagulant effect via thrombin inhibition with a Ki value of 380nM as well as by binding to pro-coagulant phospholipids of plasma. In ex-vivo conditions, RVAPLA2 (1.0μM) was non-hemolytic and non-cytotoxic to mammalian cells. It did not inhibit the collagen-induced aggregation of platelets. RVAPLA2 at a dose of 5mg/kg was not lethal to mice after 48h of injection. It demonstrated invivo anticoagulant activity possibly due to targeting thrombin and binding with plasma phospholipids.

Comparative toxicity of low dose tributyltin chloride on serum, liver, lung and kidney following subchronic exposure

Tributyltin (TBT) pollution is rampant worldwide and is a growing threat due to its bio-accumulative property. Isolated studies of TBT toxicity on different organs are available but consolidated information is greatly lacking. We planned this study to delineate the effect of subchronic (1month) exposure to low dose TBT-chloride (TBTC) (1 and 5mg/kg) in male Wistar rats. Total tin concentration was found to be significantly increased in liver, kidney and blood, and marginally in lungs. Organo-somatic indices were seen to be altered with little effect on serum biochemical markers (liver and kidney function, and general parameters). Reactive oxygen species but not lipid peroxidation content was observed to be significantly elevated both in the tissues and serum. TBTC was found to act as a hyperlipidemic agent and it also affected heme biosynthetic pathway. Hematological analysis showed that TBTC exposure resulted in minor alterations in RBC parameters. Histological studies demonstrated marked tissue damage in all the 3 organs. Calcium inhibitors (BAPTA-AM, EGTA) and antioxidants (NAC, C-PC) significantly restored TBTC induced loss in cell viability, under ex-vivo conditions. Antioxidants were evidently more efficient in comparison to the calcium inhibitors, implying major role of oxidative stress pathways in TBTC toxicity.

Small Portable Interchangeable Imager of Fluorescence for Fluorescence Guided Surgery and Research

Fluorescence guided surgery (FGS) is a developing field of surgical and oncologic research. Practically, FGS has shown useful applications in urologic surgery, benign biliary surgery, colorectal cancer liver metastasis resection, and ovarian cancer debulking. Most notably in in cancer surgery, FGS allows for the clear delineation of cancerous tissue from benign tissue. FGS requires the utilization of a fluorescent contrast agent and an intraoperative fluorescence imaging device (IFID). Currently available IFIDs are expensive, unable to work with multiple fluorophores, and can be cumbersome. This study aims to describe the development and utility of a small, cost-efficient, and interchangeable IFID made from commercially available components. Extensive research was done to design and construct a light-weight, portable, and cost-effective IFID. We researched the capabilities, size, and cost of several camera types and eventually decided on a near-infrared (NIR) charged couple device (CCD) camera for its overall profile. The small portable interchangeable imager of fluorescence (SPIIF) is a "scout" IFID system for FGS. The main components of the SPIIF are a NIR CCD camera with an articulating light filter. These components and a LED light source with an attached heat sink are mounted on a small metal platform. The system is connected to a laptop by a USB 2.0 cable. Pixielink © software on the laptop runs the system by controlling exposure time, gain, and image capture. After developing the system, we evaluated its utility as an IFID. The system weighs less than two pounds and can cover a large area. Due to its small size, it is easily made sterile by covering it with any sterile plastic sheet. To determine the system's ability to detect fluorescent signal, we used the SPIIF to detect indocyanine green under ex and in-vivo conditions and fluorescein under ex-vivo conditions. We found the SPIIF was able to detect both ICG and fluorescein under different depths of a semi-opaque colloid. Second, we found that a concentration as low as 0.5 g/ml of indocyanine green dissolved in plasma was detectable. Lastly, in a murine and human cancer model, the SPIIF was able to detect indocyanine green signal within tumors and generate a signal-to-background ratio (SBR) of 3.75. This study shows that a low-cost IFID can be made from commercially available parts. Second, this IFID is capable of in and ex-vivo detection of multiple fluorophores without sacrificing its small size or favorable ergonomics.

Three-Dimensional Laparoscopic Imaging Improves Surgical Performance on Standardized Ex-Vivo Laparoscopic Tasks

Conventional two-dimensional (2D) laparoscopy has been limited by the lack of depth perception and spatial orientation. This disadvantage may affect surgical performance, operative time, or morbidity. Depth perception, spatial resolution, and accuracy may be improved by three-dimensional (3D) vision. This study was designed to evaluate the effect of 3D imaging on the performance of novice and experienced laparoscopic surgeons executing standardized laparoscopic tasks in an ex-vivo setting. A 3D imaging system with a digital 3D full high definition (HD) and a 2D imaging with a conventional HDTV camera were used. A total of 10 laparoscopic experts and 10 novices were evaluated while performing standardized skill tasks in a pelvic trainer. Participants were divided into two groups working either with 2D or 3D imaging. The parameters measured were time until completion of each skill and total procedure time, number of losses of working material, number of missed grasps, and number of needed support. Statistical analysis was performed using the Wilcoxon test. The results showed a significant difference in favor of the 3D system for the amount of missed grasps in the experts as well as the novice group (P<0.0001). A slightly significant difference was also seen for the loss of working materials (P=0.0381 and P=0.0693). The study is limited by its small sample size. The 3D imaging system significantly improves spatial orientation and depth perception during laparoscopy under ex-vivo conditions.

Wall-to-lumen ratio of retinal arterioles

Wall-to-lumen ratio of retinal arterioles

847 3D-LAPAROSCOPIC IMAGING IMPROVES SURGICAL PERFORMANCE ON STANDARDIZED LAPAROSCOPIC TASKS

You have accessJournal of UrologyTechnology & Instruments: Laparoscopy - Malignant Disease1 Apr 2012847 3D-LAPAROSCOPIC IMAGING IMPROVES SURGICAL PERFORMANCE ON STANDARDIZED LAPAROSCOPIC TASKS Patrick Honeck, Gunnar Wendt-Nordahl, Jens Rassweiler, and Thomas Knoll Patrick HoneckPatrick Honeck Sindelfingen, Germany More articles by this author , Gunnar Wendt-NordahlGunnar Wendt-Nordahl Sindelfingen, Germany More articles by this author , Jens RassweilerJens Rassweiler Heilbronn, Germany More articles by this author , and Thomas KnollThomas Knoll Sindelfingen, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.939AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Laparoscopy has been limited by the lack of depth perception and spatial orientation by using two-dimensional imaging. This directly affects surgical performance, operative time, and morbidity. Three-dimensional vision offers the advantage of improved depth perception and accuracy. This study was designed to evaluate the effect of 3-dimensional imaging on the performance of novice and experienced laparoscopic surgeons executing standardized laparoscopic tasks in an ex-vivo setting. METHODS A 3-dimensional imaging system with a digital 3D full HD (Einstein system, Schölly Fiberoptics, Germany) and a 2-dimensional imaging with a conventional HDTV camera were used. A total of 10 laparoscopic experts and 10 novices were evaluated while performing standardized skill tasks in a pelvic trainer. Participants were devided into 2 groups working either with 2-dimensional or 3-dimensional imaging. The parameters measured were time until completion of each skill and total procedure time, number of losses of working material, number of missed grasps and number of needed support. Statistical analysis was performed using the Wilcoxon test. RESULTS The results showed s significant difference in favor of the 3-dimensional system for the amount of missed grasps in the experts as well as the novice group (p<0.0001). A slightly significant difference was also seen for the loss of working materials (p=0.0381 and p=0.0693). The study is limited by its small sample size. CONCLUSIONS The Einstein 3-dimensional imaging system sigificantly improves spatial orentation and depth perception during laparoscopy under ex-vivo conditions. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e345 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Patrick Honeck Sindelfingen, Germany More articles by this author Gunnar Wendt-Nordahl Sindelfingen, Germany More articles by this author Jens Rassweiler Heilbronn, Germany More articles by this author Thomas Knoll Sindelfingen, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Different stereoselective inhibition of monoamine oxidase-B by the R- and S-enantiomers of MD 780236.

MD 780236, a selective inhibitor of the B form of MAO behaves as an irreversible inhibitor in in-vitro conditions and mainly as a short-acting inhibitor in ex-vivo experiments. The enantiomer with the R absolute configuration (MD 240928) is fully reversible in ex-vivo conditions, whereas the S-enantiomer (MD 240931) has kept the irreversible component of the inhibition seen with MD 780236. The corresponding racemic alcohol derivative (MD 760548) is also a short-acting inhibitor of the B form of MAO; its S-enantiomer, which has an absolute configuration corresponding to that of MD 240928, has a selectivity towards the B form superior to that of the other enantiomer. The mechanism of the MAO inhibition by MD 780236 is discussed.

Investigation of the penetration behaviour of mycophenolate mofetil from a semisolid formulation into human skin ex-vivo.

Mycophenolate mofetil, the morpholinoethylester of mycophenolic acid, is an immunosuppressant used in combination with ciclosporin (cyclosporin) and corticosteroids to prevent organ rejection after heart and kidney transplantations. The drug seems also to be effective in dermal diseases after systemic administration. However, up to date mycophenolate mofetil can be only systemically administered and this is associated with several side effects such as nausea, leucopenia, sepsis, and diarrhoea. The aim of this study was to develop a topical formulation containing mycophenolate mofetil and to investigate in-vitro release and penetration into human skin ex-vivo. HPLC was applied to quantify mycophenolate mofetil after release studies from semisolid formulations using a dodecanol-collodion membrane as a lipophilic acceptor. Penetration studies with an amphiphilic cream using excised human breast skin were carried out in Franz-type diffusion cells. Mycophenolate mofetil and its active metabolite mycophenolic acid were detected by HPLC-MS after microsectioning in different skin layers. In this study the penetration of mycophenolate mofetil from an amphiphilic cream into excised human skin was shown. Additionally, the enzymatic hydrolysis of penetrated mycophenolate mofetil into mycophenolic acid was proven even under ex-vivo conditions. In-vivo a higher extent of metabolism of mycophenolate mofetil to mycophenolic acid would be expected because of the complete enzyme activity. This topical formulation might be a promising alternative to the usual systemic administration of mycophenolate mofetil in the treatment of skin diseases such as psoriasis.

Cord blood: from garbage to life-saving source.

Cord blood: from garbage to life-saving source.

CarisolvTM: an alternative to NaOCl in immature root canals?

To test the null hypothesis that Carisolv is no more effective than 1% sodium hypochlorite in cleaning uninstrumented, immature root canals. A total of 240 uniform, immature ovine incisors were decoronated at the CEJ level and randomly divided into four groups of 60. After gross pulp extirpation, canals were flooded with normal saline (negative control), 1% NaOCl, Carisolv or 5% NaOCl (positive control) and incubated for 10 min (group 1), 20 min (group 2), 30 min (group 3) or 30 min, refreshing irrigant at 10 and 20 min (group 4). SEM photomicrographs of canal wall debris in the apical, middle and coronal thirds were scored against a 5-point scale. Internal consistency was assessed by kappa statistics. Debris scores for different irrigant regimes at different canal levels were analysed by non-parametric tests (P < 0.05). Canals were consistently cleaner in the coronal and middle than apical thirds. NaOCl (5%) was consistently most effective. Carisolv and NaOCl (1%) were no more effective than normal saline in group 1 (P > 0.05), but significantly more effective than normal saline in groups 2 (middle and apical 1/3), 3 and 4 (P < 0.05). Carisolv and NaOCl (1%) had comparable activity in groups 1, 2 (middle and apical thirds) and 3, but NaOCl (1%) was significantly more effective than Carisolv in group 4 (coronal and middle thirds). 1. The ovine incisor model presents opportunities to investigate irrigation regimes under controlled ex-vivo conditions. 2.NaOCl (5%) remains the most effective irrigant for rapid debris removal in immature root canals. 3. Carisolv cleans pulp debris from the walls of immature root canals as effectively as NaOCl (1%) during static, unrefreshed wall contact for between 20 and 30 min. 4. Refreshment of NaOCl (1%) enhances its cleaning ability above that of Carisolv.

Optimization of ex-vivo expansion conditions with different growth factor combinations and concentrations maintaining long-term engrafting ability of umbilical cord blood CD34+ stem cells

Optimization of ex-vivo expansion conditions with different growth factor combinations and concentrations maintaining long-term engrafting ability of umbilical cord blood CD34+ stem cells

AN EX-VIVO EVALUATION OF VASCULAR CATHETERS FOR CONTINUOUS HEMOFILTRATION

Objectives: To measure outflow and inflow hydraulic resistance in double-lumen catheters used for hemofiltration under standardized laboratory conditions. Setting: ICU Laboratory of tertiary unit. Methods: Heparinized spent red cells diluted in polygeline solution to a constant hematocrit of 32% at 37°C were pumped using a standard Prisma M60 circuit through several hemofiltration catheters. Blood pump speed was increased and decreased in steps of 30 mL/min (30, 60, 90, 120, 150, and 180 mL/min) and catheter outflow and inflow pressures recorded and used to define the pressure flow relationship (line of hydraulic resistance) for each. Results: Double-lumen catheters posed different resistances to outflow or inflow. Among the <15 cm long catheters, the 11.5 Fr Quinton-Mahurkar (0.56 mmHg/mL/min) catheter offered the least resistance to outflow, while the Medcomp 11.5 Fr catheter offered the least resistance to inflow (0.78 mmHg/mL/min). Among the >19 cm long catheters, the 13.5 Fr Vascath Niagara catheter showed the lowest blood flow resistance to both outflow (0.63 mmHg/mL/min) and inflow (0.83 mmHg/mL/min). Longer catheters did not pose statistically greater resistance to both outflow and inflow. Resistance to inflow was consistently greater than resistance to outflow (p = 0.003). Overall, the Prisma M60 blood circuit alone accounted for 40% of the total extracorporeal circuit blood flow resistance. Conclusions: Proprietary hemofiltration catheters have variable resistance to blood flow under standard ex-vivo conditions. This ex-vivo information might be useful to clinicians in guiding their choice of catheters for clinical use.

Diagnosis of acute lung embolism with spiral CT and 3D reconstruction. Development of an animal model and technical probe in an ex-vivo experiment

To develop a model for simulation the CT morphologic situation of acute pulmonary embolism, to evaluate the accuracy of spiral CT and 3D reconstruction in the detection of artificial emboli and to investigate the influence of the orientation of emboli depending on z-axis orientation. Standardized artificial emboli made of wax and of defined size and shape were positioned into the pulmonary arteries of porcine lungs. Castings of the embolized pulmonary arterial trees were made by injection of a special opaque resin. After performance of spiral CT the data sets of the emboli and the pulmonary arteries were post-processed. The 3D segmentations were compared with the anatomic preparation to evaluate the accuracy of spiral CT/3D reconstruction-technique. Technical specimens simulating CT-morphology of acute embolized vessels underwent spiral CT in six different positions with respect to the z-axis. The CT data were reconstructed using a standardized and a contrastadapted method with interactive correction. The 3D emboli were analysed under qualitative aspects, and measurements of their extent were done. In nearly 91%, there was complete agreement between CT and the corresponding findings at the anatomical preparation. Measurements of the 3D reconstructed technical specimens showed discrepancies of shape and size in dependence of the size of the original preparation, orientation and reconstruction technique. Overestimation up to 4 mm and underestimation to 2.2 mm were observed. Measurements of preparations with heights from 14 to 26 mm showed variances of +/- 1.5 mm (approximately 6-11%). The presented models are suitable to simulate CT morphology of acute pulmonary embolism under ex-vivo conditions. Accuracy in the detection of artificial emboli using spiral CT/3D reconstruction is affected by localization, size and orientation of the emboli and the reconstruction technique.