In vitro assessment of a collagen sponge for engineering urothelial grafts

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Abstract
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. Collagen sponges were exposed to a continuous flow of urine at pH 5.3 and 6.3 for 1 week. The sponges were examined microscopically for crystal deposition and analysed for their calcium content. Two cell lines, RT112, derived from a well-differentiated transitional cell carcinoma, and UROtsa, an immortalized urothelial cell line, were seeded on the collagen sponges. Cells were cultured for 6, 12 and 21 days. The pattern of growth was analysed by histology and immunostaining with a pan-cytokeratin antibody. Growth was assayed to quantify cell proliferation on the sponges. No crystals were evident on any of the collagen sponges. Calcium deposition 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 at 5–10 days. The sponge remained easy to handle after 3 weeks in culture. 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.