Expression of components of the urothelial cholinergic system in bladder and cultivated primary urothelial cells of the pig.

BACKGROUND: Porcine urinary bladders are widely used for uro-pharmacological examinations due to their resemblance to the human organ. However, characterisations of the porcine urothelium at the molecular level are scarce up to now. As it has become clear over the last years that this tissue plays an important role in the signaling-pathways of the bladder, we examined whether the transporter and receptor pattern (with focus on the transmitter acetylcholine) is comparable to the human urothelium. With regard to in vitro studies, we also investigated if there is a difference between the native tissue and cultivated primary urothelial cells in culture. METHODS: Urothelium from German Landrace and Göttingen Minipig bladders was collected. One part of the German Landrace tissue was used for cultivation, and different passages of the urothelial cells were collected. The actual mRNA expression of different transporters and receptors was examined via quantitative real-time PCR. These included the vesicular acetylcholine transporter (VAChT), the choline acetyl transferase (ChAT), organic cation transporters 1-3 (OCT1-3), organic anion transporting polypeptide 1A2 (OATP1A2), P-glycoprotein (ABCB1), the carnitine acetyl-transferase (CarAT), as well as the muscarinic receptors 1-5 (M1-5). RESULTS: There is a strong qualitative resemblance between the human and the porcine urothelium with regard to the investigated cholinergic receptors, enzymes and transporters. CarAT, OCT1-3, OATP1A2 and ABCB1 could be detected in the urothelium of both pig races. Moreover, all 5 M-receptors were prominent with an emphasis on M2 and M3. VAChT and ChAT could not be detected at all. Cultures of the derived urothelial cells showed decreased expression of all targets apart from ABCB1 and CarAT. CONCLUSIONS: Based on the expression pattern of receptors, transporters and enzymes of the cholinergic system, the porcine urinary bladder can be regarded as a good model for pharmacological studies. However, cultivation of primary urothelial cells resulted in a significant drop in mRNA expression of the targets. Therefore, it can be concluded that the intact porcine urothelium, or the whole pig bladder, may be appropriate models for studies with anticholinergic drugs, whereas cultivated urothelial cells have some limitation due to significant changes in the expression levels of relevant targets.

Evaluation of electrical impedance tomography for determination of urinary bladder volume: comparison with standard ultrasound methods in healthy volunteers.

BACKGROUND: Continuous non-invasive urinary bladder volume measurement (cystovolumetry) would allow better management of urinary tract disease. Electrical impedance tomography (EIT) represents a promising method to overcome the limitations of non-continuous ultrasound measurements. The aim of this study was to compare the measurement accuracy of EIT to standard ultrasound in healthy volunteers. METHODS: For EIT of the bladder a commercial device (Goe MF II) was used with 4 different configurations of 16 standard ECG electrodes attached to the lower abdomen of healthy participants. To estimate maximum bladder capacity (BCmax) and residual urine (RU) two ultrasound methods (US-Ellipsoid and US-L × W × H) and a bedside bladder scanner (BS), were performed at the point of urgency and after voiding. For volume reference, BCmax and RU were validated by urine collection in a weight measuring pitcher. The global impedance method was used offline to estimate BCmax and RU from EIT. RESULTS: The mean error of US-Ellipsoid (37 ± 17%) and US-L × W × H (36 ± 15%) and EIT (32 ± 18%) showed no significant differences in the estimation of BCmax (mean 743 ± 200 ml) normalized to pitcher volumetry. BS showed significantly worse accuracy (55 ± 9%). Volumetry of RU (mean 152.1 ± 64 ml) revealed comparable higher errors for both EIT (72 ± 58%) and BS (63 ± 24%) compared to US-Ellipsoid (54 ± 25%). In case of RU, EIT accuracy is dependent on electrode configuration, as the Stripes (41 ± 25%) and Matrix (38 ± 27%) configurations revealed significantly superior accuracy to the 1 × 16 (116 ± 62%) configuration. CONCLUSIONS: EIT-cystovolumetry compares well with ultrasound techniques. For estimation of RU, the selection of the EIT electrode configuration is important. Also, the development of an algorithm should consider the impact of movement artefacts. Finally, the accuracy of non-invasive ultrasound accepted as gold standard of cystovolumetry should be reconsidered.

Two differentially structured collagen scaffolds for potential urinary bladder augmentation: proof of concept study in a Göttingen minipig model.

BACKGROUND: The repair of urinary bladder tissue is a necessity for tissue loss due to cancer, trauma, or congenital abnormalities. Use of intestinal tissue is still the gold standard in the urological clinic, which leads to new problems and dysfunctions like mucus production, stone formation, and finally malignancies. Therefore, the use of artificial, biologically derived materials is a promising step towards the augmentation of this specialised tissue. The aim of this study was to investigate potential bladder wall repair by two collagen scaffold prototypes, OptiMaix 2D and 3D, naïve and seeded with autologous vesical cells, as potential bladder wall substitute material in a large animal model. METHODS: Six Göttingen minipigs underwent cystoplastic surgery for tissue biopsy and cell isolation followed by implantation of unseeded scaffolds. Six weeks after the first operation, scaffolds seeded with the tissue cultured autologous urothelial and detrusor smooth muscle cells were implanted into the bladder together with additional unseeded scaffolds for comparison. Cystography and bladder ultrasound were performed to demonstrate structural integrity and as leakage test of the implantation sites. Eighteen, 22, and 32 weeks after the first operation, two minipigs respectively were sacrificed and the urinary tract was examined via different (immunohistochemical) staining procedures and the usage of two-photon laser scanning microscopy. RESULTS: Both collagen scaffold prototypes in vivo had good ingrowth capacity into the bladder wall including a quick lining with urothelial cells. The ingrowth of detrusor muscle tissue, along with the degradation of the scaffolds, could also be observed throughout the study period. CONCLUSIONS: We could show that the investigated collagen scaffolds OptiMaix 2D and 3D are a potential material for bladder wall substitution. The material has good biocompatible properties, shows a good cell growth of autologous cells in vitro, and a good integration into the present bladder tissue in vivo.

Telemetric monitoring of bladder function in female Göttingen minipigs.

OBJECTIVES: To generate real-time radio-telemetric urodynamic reference data of maximum detrusor pressure (Pdet max ), maximum flow rate (Qmax) and estimated grade of infravesical obstruction, as well as duration of detrusor contraction (DOC), in female Göttingen minipigs and to describe translational aspects of the use of Göttingen minipigs for urological research. MATERIALS AND METHODS: A telemetric transmitter was implanted into five female Göttingen minipigs, and 24 h measurements in metabolic cages were taken. Through operator-based analysis, synchronized real-time radio-telemetric cystometric data with flowmetric data and video sequences were used to determine voiding detrusor contractions (VCs) and non-voiding detrusor contractions (NVCs). Furthermore, data from telemetric natural-filling cystometry from free-moving and restricted maintenance conditions were compared for potential differences. RESULTS: The median (range) Pdet max of VCs was 120.6 (21.0-370.0) cmH2 O and, therefore, significantly different from that of NVCs (64.60 [20.4-280.6 cmH2 O] cm H2 O). Intraindividual comparison of minipig data revealed great differences in voiding contractions. The effects of limited movement on VCs were analysed and showed significantly higher Pdet max and lower DOC than in free-moving conditions. CONCLUSION: The presented data can be used for the development of telecystometric implanted minipig models, to investigate changes of detrusor function such as under- or overactivity, and might serve as model for bladder changes occurring with iatrogenic bladder outlet obstruction (BOO) or different therapeutic options for overactive bladder. Radio-telemetric real-time natural filling and voiding cystometries are feasible, reproducible in non-anaesthetized minipigs with free or limited movement and can give new insights into circadian behaviour and physiological and pathological bladder function.

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.

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.