180 Primary human urothelial cell culture on a matrix of spider silk: A novel technique in bladder reconstruction

Abstract

Various diseases of the genitourinary tract can cause damage to the bladder and result in urinary retention or incontinence. Treatment options of affected bladders include augmentation cystoplasty, in which the bladder is enlarged by using a piece of intestine. Since the use of intestine in the urinary tract can cause serious adverse effects, new (bio)materials are being tested for bladder reconstruction. However, the ideal (bio)material for reconstruction of the bladder has not yet been discovered, mostly due to mechanical failure in vivo. A promising natural biomaterial is spider silk, which is outstanding in its slow degradation rate and mechanical properties. In this study, we assess the in vitro potential of spider silk as a suitable biomaterial for bladder reconstruction by cultivating primary human urothelial cells (HUCs) on native spider silk matrices. Here, we manufactured dental wire frames which were woven with the dragline silk of female Nephila Clavipes spiders under standardized conditions. Upon sterilization of spider silk frames, HUCs were cultivated on spider silk matrices and evaluated for adhesion, expansion, viability and cellular differentiation. Analysis of cell morphology and filamentous actin in HUCs revealed abundant adhesion sites and bridging of HUCs to spider silk matrices. In addition, qRT-PCRbased analysis of adhesion molecules present in HUCs revealed down-regulation of certain adhesion molecules when HUCs were cultured on spider silk. Expansion of HUCs on spider silk matrices was determined over time using Presto Blue and DAPI cell count in fields of vision and showed that spider silk supports expansion of HUCs. In vitro biocompatibility of spider silk was assessed by culturing HUCs in an extract of spider silk and revealed no cytotoxic effects on HUCs. This is supported by live/dead staining which demonstrated that HUCs were viable when cultured on spider silk matrices. QRT-PCR-based analysis of epithelial-mesenchymal transition, fibrosis and cellular differentiation markers expressed in HUCs when cultured on spider silk showed, besides down-regulation of CD44, no differences compared to control. Flow cytometry-based analysis of urothelial cellular differentiation markers CD90, CD44 and CD49f demonstrated a small subpopulation lower in CD44 expression at protein level when HUCs were cultured on spider silk. These results indicate that spider silk causes HUCs to mature slightly faster, but has no major effects on cellular differentiation of HUCs. Together, results describe a novel biomaterial which supports adhesion, expansion and viability of HUCs. Our study gives new understanding of cellular mechanisms occurring in HUCs when cultured on spider silk and demonstrates that spider silk is a promising biomaterial for bladder reconstruction.