Introduction: Human amniotic membrane (AM) is the innermost layer of placenta and is composed of three distinct strata, namely single epithelial layer, a thick basement membrane and avascular stroma. AM has been widely adopted as a biomaterial in tissue engineering and surgical reconstructions, since it promotes wound healing and epithelialization, has low immunogenicity, reduces inflammation and scarring, and was also shown to possess anti-cancer properties (1). We have shown that AM promotes formation of highly differentiated urothelium, in particular when urothelial cells are cultured on AM stromal side (2). Although AM stroma scaffold allows establishment of differentiated urothelium, its orientation probably does not favour AM integration into surrounding tissue, if applied in vivo. Therefore, we established an in vitro AM model which would allow incorporation of fibroblasts into the AM stroma as well as promote urothelial differentiation.

Materials and Methods: Porcine bladder fibroblasts were seeded onto porous membranes and were labelled with green lipophilic dye when subconfluent. Cryopreserved human AMs mounted into Scaffadex holders were de-epithelized and laid onto cultures of fibroblasts in such a manner that fibroblasts were in close contact with the AM stromal layer. Fibroblasts were left to grow into AM stroma in medium adapted for fibroblasts for up to a week, when porcine urothelial cells were seeded on the opposite side of the AM scaffold i.e. on the AM basement membrane. The constructs were maintained for three additional weeks in the medium adapted for urothelial cells. As a control, urothelial cells seeded on the de-epithelized AM scaffold without integrated fibroblasts were used to evaluate contribution of fibroblasts on urothelial differentiation. Growth of urothelial cells was monitored daily. Integration of fibroblasts and differentiation of urothelial cells was determined by electron microscopy, histological and immunofluorescence techniques.

Results: Fibroblasts cultured in close contact with AM stroma in the medium adapted for fibroblasts integrated into AM stroma and remained viable after three additional weeks of maintenance in the medium for urothelial cells. Proliferation and differentiation of urothelial cells depended largely on the presence of fibroblasts. In comparison to the control, urothelial cells on fibroblast enriched AM scaffolds reached confluency faster, and more importantly, reached higher differentiation stage (Figure 1).

Discussion and Conclusion: Our data demonstrate that bladder fibroblast enriched AM scaffold promotes urothelial differentiation in vitro. Besides being suitable in vitro research tool to study epithelial-mesenchymal interactions, this model also indicates that de-epithelized human AM could integrate into bladder lamina propria and could be as such potentially used as a graft for urothelial reconstruction.

References

1. A. C. Mamede et al., Amniotic membrane: from structure and functions to clinical applications. Cell Tissue Res 349, 447, 2012.

2. U.D. Jerman et al., Amniotic membrane scaffolds enable the development of tissue-engineered urothelium with molecular and ultrastructural properties comparable to that of native urothelium. Tissue Eng Part C Methods 20, 317, 2014.

Figures:

Figure 1: (A, B) Scanning electron microscopy. Porcine urothelial cells cultured on de-epithelized AM scaffolds enriched with porcine bladder fibroblasts (A) established highly differentiated urothelium, whereas those maintained on AM scaffolds without integrated fibroblast (B) differentiated partially. Scale bars: 10 µm.

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EMC Abstracts - https://emc-proceedings.com/abstract/amniotic-membrane-scaffold-enriched-with-bladder-fibroblasts-promotes-establishment-of-highly-differentiated-urothelium/