Hypothesis / aims of study
For urethral reconstruction in urethral stricture or hypospadias, over 300 surgical techniques are known. A wide variety of grafts, including autologous skin and oral mucosa, have been used. However, these substitutes are inferior to native urethral tissue and can lead to (recurrent) stricture and/or graft failure.
Tissue engineering may be a solution for restoration of the urethra in complex stricture or hypospadias cases. This project will focus on the luminal epithelial cells. Previous research has revealed that urethra epithelium is different from bladder epithelium [1]. However, as urethral biopsies to extract urethral cells may lead to stricture formation, bladder cells may be an alternative in urethral tissue engineering.
Epithelium in the urethra is exposed to fluid flow, unlike bladder epithelium, were fluid is more constantly present. Biomechanical cues influence the behavior and differentiation of cells, and our hypothesis is that fluidic flow induced shear stress may stir cells towards different organization. To investigate whether bladder epithelial cells may be used as a substitute for more difficult to obtain urethra epithelial cells for tissue engineering purposes, we compared the cell response to fluidic flow induced shear stress, comparing urethral epithelial cells with bladder epithelial cells.
Study design, materials and methods
Primary cells from both bladder and urethra were isolated from male pigs [2]. Microfluidic devices were used to study effects of shear stress. Pump driven systems (ibidi GmbH, Gräfelfing, Germany) were compared with the F300R microfluidic device (Finnadvance, Oulo, Finland) on the rocking system (WAVE) (FinnAdvance, Oulo, Finland). The bladder and urethra derived epithelial cells were exposed for 72 hours to flow, causing fluidic shear stress (FSS) of τmax= 10.0 dyn/cm2 and τmax= 20.0 dyn/cm2 in a ibidi flow system.
The F300R microfluidic device was placed on the platform of the rocking machine with the following settings: set timer: 0,00 min; set hold time: 0,29 min; set speed: 30 rpm; and set angle: -18,43º / 18,43º. These settings together with a volume of 240 μL for each channel, results in a FSS of 0,049 dyne/cm2, which is the maximum FSS that the set-up consisting of the F300R microfluidic device and the rocker can generate.
For both systems, cell elongation, cell alignment and actin fiber alignment were analyzed.
Interpretation of results
Both bladder and urethral epithelial cells similarly adapted to FSS: they both elongate and align in the direction of the flow. So in this respect, both cell types could potentially be used in tissue engineering for urethral reconstruction. This is important information, because the harvesting of bladder epithelium is much easier than of urethra epithelium: bladder epithelial cells can be isolated from urine or bladder washout or may be obtained by biopsy, in contrast to urethral epithelial cells. Next steps in our approach would be creating intermitted flow to mimic natural voiding in patients as well as comparing other sources of cells (like oral keratinocytes) to these urogenital epithelial cells.