In vitro assessment of a collagen sponge for engineering urothelial grafts

Objective To investigate the deposition of urinary crystals and the growth characteristics of urothelial cells on a collagen sponge, as a preliminary step in engineering urothelial autologous grafts. Materials and methods Collagen sponges were exposed to a continuous flow of urine at pH 5.3 and 6.3 for 1 week, The sponges were examined microscopica lly for crystal deposition and analysed for their calcium content. Two cell lines, RT112, derived from a well-differentiated transitional cell carcino ma, and UROtsa, an immortalized urothelial cell line, were seeded on the co llagen sponges. Cells were cultured for 6, 12 and 21 days. The pattern of g rowth was analysed by histology and immunostaining with a pan-cytokeratin a ntibody. Growth was assayed to quantify cell proliferation on the sponges, Results No crystals were evident on any of the collagen sponges. Calcium de position was negligible at pH 5,3. Although calcium levels were measurable at pH 6,3, the levels were very low, Both cell lines attached and grew in a stratified manner on the collagen sponge, RT112 forming a layer 6-8 cells thick, and UROtsa a layer 4-6 cells thick; cell proliferation was maximal a t 5-10 days. The sponge remained easy to handle after 3 weeks in culture, Conclusion These findings show that collagen sponges support the growth and stratification of urothelial cells, and indicate that the collagen sponge is a suitable substrate for developing urothelial autologous grafts.