Hypothesis / aims of study
In males, a large part of the urethra is surrounded by the vascular corpus spongiosum, providing the urethra with nutrients and protection. In current reconstructive surgery for hypospadias or urethral stricture disease, the urethra is created from epithelial buccal mucosa or foreskin, without any support of the corpus spongiosum. There is no autologous tissue available mimicking the corpus spongiosum. Addition of spongious tissue could promote healing and prevent complications. Although it is not possible yet to recreate spongious tissue, we are trying to set the first steps.
For tissue to grow, extracellular matrix (ECM) is an essential non-cellular component of the microenvironment of cells, comprised of a network of macromolecules including polysaccharide glycosaminoglycans (GAGs) and proteins including collagens, laminins, and fibronectin [1]. For tissue engineering purposes, hydrogels with different compositions are used to grow cells. The aim of this study is to generate hydrogels from decellularized native urethral and spongious tissue ECM combined with silk fibroin in different proportions, for support. In this hydrogel, microvessel forming cells can be incorporated and epithelial cells can be seeded on top. Our ultimate goal is to create a combination of a vascular corpus spongiosum with functional mucosa to restore the urethra.
Study design, materials and methods
Tissue was obtained from deceased horses that had been donated to science by their owners in the veterinary hospital (waste material). From fresh corpses, the penis was dissected and transported to our institute, where it was frozen until further dissection. The urethra and corpus spongiosum were isolated and decellularized using sodium dodecyl sulfate, followed by pepsin digestion. Equine dECM was DNA quantified and important ECM proteins like collagens, elastin and fibrinogen were indicated through western blot. Gels were tested on their sol fraction and swelling ratio and were taken into in vitro cell assays and in vivo Chick Chorioallantoic Membrane (CAM) assays.
Results
The isolated horse corpus spongiosum hydrogel showed low DNA content and abundance of ECM proteins as expected. The 1:1 (dECM : silk fibroin) hydrogels exhibited the lowest sol fraction and swelling ratio. In general, the hydrogels showed heterogenic crosslinking and high auto-fluorescence, which resulted in difficult analysis. De hydrogels supported cell growth and cells were not able to grow on silk fibroin only, missing dECM (figure 1). Urothelium and urethra epithelium showed less stress fibers when growing on dECM coated coverslips, compared to gelatin. All the hydrogels grafted on the CAM showed survival of the chick embryos and CAM vessels grow towards the hydrogel (figure 2). For both total vessel length and vasculogenic index, the dECM : silk fibroin hydrogel showed higher, although not significantly different, counts compared to gelatin control gels.
Interpretation of results
Cells display less stress fibers when grown on ECM coating compared to gelatin, indicating the created niche is favored by the cells. In the CAM assay, the vessels of the CAM grow towards the hydrogel, showing vasculogenic properties of the hydrogel. Unfortunately, biomechanical properties were hard to control, and the gels were highly auto fluorescent, making them hard to analyse.