Tissue Engineering des Harnleiters: Entwicklung eines Bioreaktorsystems und die anschließende Charakterisierung der gezüchteten Biohybride

Heutzutage betreffen 1-2,5% der Verletzungen des Urogenitaltraktes die Harnleiter und führen dort zu ernsthaften Beeinträchtigungen der Harnableitung. Die derzeit klassischen rekonstruktiven Operationstechniken sind zwar in der Lage bis zu 15 cm eines geschädigten Harnleiters wieder herzustellen, jedoch ist dies von der Position der Verletzung abhängig. Eine aufwendigere Technik, mit einem Darminterponat, welches deutlich längere Strecken überbrücken kann, ist auch mit postoperativen Begleiterscheinungen behaftet. Zu diesen Begleiterscheinungen zählen unter anderem Verengungen, Kalzifizierungen, oder auch übermäßige Schleimproduktion. Die vorliegende Dissertation präsentiert einen neuen Ansatz zum Harnleiterersatzes mit Hilfe des Tissue Engineerings. Zu diesem Zweck wurde ein Bioreaktorsystem entwickelt und mit verschiedenen Trägermaterialien und Zellen getestet. Die hergestellten Biohybride bestanden aus einem natürlichen Trägermaterial (Kollagen) oder einem Kompositmaterial (Fibrin und PVDF). Dieses wurden in verschieden Kombinationen mit verschiedenen Zelltypen (Urotsa, C2C12, Fibroblasten und/oder glatten Muskelzellen) kultiviert. Zudem wurde ein mechanischer Stimulationsmechanismus zur Ausrichtung der aufgebrachten Muskelzellen etabliert. Das Bioreaktorsystem zeigt seine Zuverlässigkeit bei der Kultivierung von Zellen in einem modular aufgebauten Bioreaktor von bis zu 2 Wochen. Die wichtigsten Kultivierungsparameter (Temperatur, pH-Wert und CO2-Konzentration) schwanken nur leicht um den Sollwert mit einer Standardabweichung von 1 °C, 0,2 (pH) und 0,3% (CO2-Konzentration). Des Weiteren zeigt das System, dass Kokultivierungen von Urotsa und C2C12 Zellen in Kombination mit einem porösen Kollagenmaterial (Optimaix 3D) möglich sind. Das Trägermaterial ist dabei nahtlos und verfügt über radial verlaufende, miteinander vernetzte Poren mit lokal variierenden Durchmessern. Das System der Mechanotransduktion baut auf einem Kyphoplastiekatheter auf und wurde an primären glatten Muskelzellen angewendet, die sich in einer Fibrinmatrix befanden. Diese Matrix wurde durch ein PVDF-Netz stabilisiert. Mechanisch stimulierte und nichtstimulierte Biohybride wurden biochemisch und mechanisch untersucht, wobei keine signifikanten Unterschiede in Bezug auf ihre Wirkung als Diffusionsbarriere (gegen Harnstoff und Kreatinin) oder der Reißfestigkeit festgestellt werden konnte. Die mechanische Stimulation führte jedoch zur Ausrichtung von glatten Muskelzellen in longitudinaler (außen) und zirkulärer (innerhalb der Fibrinmatrix) Richtung. Dies wurde durch histologische Auswertungen bestätigt. Die Ergebnisse zeigen, dass das Bioreaktorsystem aufgrund des Stimulationsmechanismus in der Lage ist, Biohybride mit zwei verschiedenen Zellausrichtungen zu generieren. Das Potential des gezüchteten Gewebes muss in weiteren Untersuchungen, u.a. Tierversuchen, charakterisiert werden.Today, 1-2.5% of all genitourinary tract trauma affect the ureters, leading to severe impairments of the urine’s drainage. Till now, classical reconstructive surgery has the possibility to bridge up to 15 cm of damaged tissue, depending on the location. Even the “gold standard”, with intestine interposition, where longer distances can be overcome, can lead to postoperative side effects like stricture formation, calcification or excessive mucous production. This thesis presents a new attempt for the replacement of damaged ureters by the use of tissue engineering technologies. A bioreactor system is developed for the generation of tubular biohybrids consisting of a natural and a composite biomaterial (pure collagen or fibrin and PVDF) in combination with different cell types (Urotsa, C2C12, fibroblasts and primary smooth muscle cells). A mechanotransduction is established for a mechanical stimulation and orientation of the applied cells. The bioreactor system allows a reliable incubation of a modularly constructed bioreactor for the cultivation of mammalian cells for up to two weeks. Incubation parameters are kept in defined ranges with deviations of ≤1 °C (temperature), ≤0.2 (pH value) and ≤0.3% (CO2 concentration). Cocultivations of Urotsa and C2C12 cells are possible in combination with a porous OPTIMAIX 3D, which has radially oriented pores with varying diameters. Mechanical stimulation that is based on a kyphoplasty catheter is applied on primary smooth muscle cells that are embedded in a PVDF-supported fibrin matrix. Biochemical and mechanical evaluations reveal that there is no significant difference between a mechanically stimulated and an unstimulated prosthesis with respect to the diffusion of urea/creatinine or the rupture pressure. Histological analysis shows two distinguishable cell layers with a longitudinal alignment on the outer surface and a circular orientation in the matrix of the stimulated fibrin-based prosthesis. It can be concluded that biohybrids can be generated in this bioreactor system with at least two different cellular orientations. These biohybrids need to be characterised in further (animal) experiments

MR Angiography of Peripheral Arterial Stents: In Vitro Evaluation of 22 Different Stent Types

Purpose. To evaluate stent lumen visibility of a large sample of different peripheral arterial (iliac, renal, carotid) stents using magnetic resonance angiography in vitro. Materials and Methods. 21 different stents and one stentgraft (10 nitinol, 7 316L, 2 tantalum, 1 cobalt superalloy, 1 PET + cobalt superalloy, and 1 platinum alloy) were examined in a vessel phantom (vessel diameters ranging from 5 to 13 mm) filled with a solution of Gd-DTPA. Stents were imaged at 1.5 Tesla using a T1-weighted 3D spoiled gradient-echo sequence. Image analysis was performed measuring three categories: Signal intensity in the stent lumen, lumen visibility of the stented lumen, and homogeneity of the stented lumen. The results were classified using a 3-point scale (good, intermediate, and poor results). Results. 7 stents showed good MR lumen visibility (4x nitinol, 2x tantalum, and 1x cobalt superalloy). 9 stents showed intermediate results (5x nitinol, 2x 316L, 1x PET + cobalt superalloy, and 1x platinum alloy) and 6 stents showed poor results (1x nitinol, and 5x 316L). Conclusion. Stent lumen visibility varies depending on the stent material and type. Some products show good lumen visibility which may allow the detection of stenoses inside the lumen, while other products cause artifacts which prevent reliable evaluation of the stent lumen with this technique

Water and sodium intake habits and status of ultra-endurance runners during a multi-stage ultra-marathon conducted in a hot ambient environment: an observational field based study

<p>Abstract</p> <p>Background</p> <p>Anecdotal evidence suggests ultra-runners may not be consuming sufficient water through foods and fluids to maintenance euhydration, and present sub-optimal sodium intakes, throughout multi-stage ultra-marathon (MSUM) competitions in the heat. Subsequently, the aims were primarily to assess water and sodium intake habits of recreational ultra-runners during a five stage 225 km semi self-sufficient MSUM conducted in a hot ambient environment (T_max range: 32°C to 40°C); simultaneously to monitor serum sodium concentration, and hydration status using multiple hydration assessment techniques.</p> <p>Methods</p> <p>Total daily, pre-stage, during running, and post-stage water and sodium ingestion of ultra-endurance runners (UER, <it>n</it> = 74) and control (CON, <it>n</it> = 12) through foods and fluids were recorded on Stages 1 to 4 by trained dietetic researchers using dietary recall interview technique, and analysed through dietary analysis software. Body mass (BM), hydration status, and serum sodium concentration were determined pre- and post-Stages 1 to 5.</p> <p>Results</p> <p>Water (overall mean (SD): total daily 7.7 (1.5) L/day, during running 732 (183) ml/h) and sodium (total daily 3.9 (1.3) g/day, during running 270 (151) mg/L) ingestion did not differ between stages in UER (<it>p</it> < 0.001 <it>vs</it>. CON). Exercise-induced BM loss was 2.4 (1.2)% (<it>p</it> < 0.001). Pre- to post-stage BM gains were observed in 26% of UER along competition. Pre- and post-stage plasma osmolality remained within normal clinical reference range (280 to 303 mOsmol/kg) in the majority of UER (<it>p</it> > 0.05 <it>vs</it>. CON pre-stage). Asymptomatic hyponatraemia (<135 mmol/L) was evident pre- and post-stage in <it>n</it> = 8 UER, corresponding to 42% of sampled participants. Pre- and post-stage urine colour, urine osmolality and urine/plasma osmolality ratio increased (<it>p</it> < 0.001) as competition progressed in UER, with no change in CON. Plasma volume and extra-cellular water increased (<it>p</it> < 0.001) 22.8% and 9.2%, respectively, from pre-Stage 1 to 5 in UER, with no change in CON.</p> <p>Conclusion</p> <p>Water intake habits of ultra-runners during MSUM conducted in hot ambient conditions appear to be sufficient to maintain baseline euhydration levels. However, fluid over-consumption behaviours were evident along competition, irrespective of running speed and gender. Normonatraemia was observed in the majority of ultra-runners throughout MSUM, despite sodium ingestion under benchmark recommendations.</p

Identification of S71-Related Human Endogenous Retroviral Sequences with Full-Length pol Genes

AbstractThe human genome contains sequences related to the simian sarcoma-associated virus SSAV. One of these endogenous retrovital elements, S71, is truncated in the pol gene and carries an insertion of a solitary HERV-K LTR. Using a PCR approach we have now identified further S71-related retroviral elements that lack the HERV-K LTR insertion and contain a full-length retroviral reverse transcriptase. Two of these sequences, pCRTK1 and pCRTK6, were cloned and further characterized. Clones pCRTK1 and pCRTK6 showed between 85 and 90% nucleotide hemology to each other and to S71 within the "tether" region of the pol gene, indicating that pCRTK1 and pCRTK6 clearly belong to the S71 subgroup of C-type-related human endogenous retroviral elements. Some point mutations inactivating the reverse transcriptase are located at the same positions in pCRTK1 and pCRTK6. Therefore, we assume that these S71-related elements were dispersed in the human genome by reintegration as defective proviruses, probably using enzymes for retrotransposition provided in trans by other retrotransposons or by cellular genes. Examination of the presence of S71-related elements in apes and Old World monkeys revealed that the deletion of reverse transcriptase sequences in S71 has occurred in the lineage of primates prior to the insertion of the HERV-K LTR

Development of a Bioreactor to Culture Tissue Engineered Ureters Based on the Application of Tubular OPTIMAIX 3D Scaffolds

Regenerative medicine, tissue engineering and biomedical research give hope to many patients who need bio-implants. Tissue engineering applications have already been developed based on bioreactors. Physiological ureter implants, however, do not still function sufficiently, as they represent tubular hollow structures with very specific cellular structures and alignments consisting of several cell types. The aim of this study was to a develop a new bioreactor system based on seamless, collagenous, tubular OPTIMAIX 3D prototype sponge as scaffold material for ex-vivo culturing of a tissue engineered ureter replacement for future urological applications. Particular emphasis was given to a great extent to mimic the physiological environment similar to the in vivo situation of a ureter. NIH-3T3 fibroblasts, C2C12, Urotsa and primary genitourinary tract cells were applied as co-cultures on the scaffold and the penetration of cells into the collagenous material was followed. By the end of this study, the bioreactor was functioning, physiological parameter as temperature and pH and the newly developed BIOREACTOR system is applicable to tubular scaffold materials with different lengths and diameters. The automatized incubation system worked reliably. The tubular OPTIMAIX 3D sponge was a suitable scaffold material for tissue engineering purposes and co-cultivation procedures.</jats:p

Mechanical induction of bi-directional orientation of primary porcine bladder smooth muscle cells in tubular fibrin-poly(vinylidene fluoride) scaffolds for ureteral and urethral repair using cyclic and focal balloon catheter stimulation

To restore damaged organ function or to investigate organ mechanisms, it is necessary to prepare replicates that follow the biological role model as faithfully as possible. The interdisciplinary field of tissue engineering has great potential in regenerative medicine and might overcome negative side effects in the replacement of damaged organs. In particular, tubular organ structures of the genitourinary tract, such as the ureter and urethra, are challenging because of their complexity and special milieu that gives rise to incrustation, inflammation and stricture formation. Tubular biohybrids were prepared from primary porcine smooth muscle cells embedded in a fibrin gel with a stabilising poly(vinylidene fluoride) mesh. A mechanotransduction was performed automatically with a balloon kyphoplasty catheter. Diffusion of urea and creatinine, as well as the bursting pressure, were measured. Light and electron microscopy were used to visualise cellular distribution and orientation. Histological evaluation revealed a uniform cellular distribution in the fibrin gel. Mechanical stimulation with a stretch of 20% leads to a circumferential orientation of smooth muscle cells inside the matrix and a longitudinal alignment on the outer surface of the tubular structure. Urea and creatinine permeability and bursting pressure showed a non-statistically significant trend towards stimulated tissue constructs. In this proof of concept study, an innovative technique of intraluminal pressure for mechanical stimulation of tubular biohybrids prepared from autologous cells and a composite material induce bi-directional orientation of smooth muscle cells by locally and cyclically applied mechanical tension. Such geometrically driven patterns of cell growth within a scaffold may represent a key stage in the future tissue engineering of implantable ureter replacements that will allow the active transportation of urine from the renal pelvis into the bladder. </jats:p