Urothlelium Facilitates the Recruitment and Trans-Differentiation of Fibroblasts into Smooth Muscle in Acellular Matrix

Abstract

Tissue engineering has shown promise for patients requiring bladder reconstruction secondary to diverse diseases such as spina bifida and tuberculosis. In this study we addressed whether urothelium in conjunction with an acellular bladder matrix is necessary for the recruitment of host cells to repopulate the graft and whether the urothelium facilitates trans-differentiation of host fibroblastic cells into bladder smooth muscle type cells. Urothelium from embryonic mice and rats (E18) was microdissected from bladder stroma after enzymatic digestion with 10 mM ethylenediaminetetraacetic acid. Acellular bladder matrix was prepared from postnatal day 1 rat and mouse pups using a combination of membrane disruption and DNA digestion. Urothelium was placed inside the bladder matrix and then grown either underneath the renal capsule or subcutaneously in athymic mice for 2 to 24 weeks. Grafts were sectioned and immunocytochemistry was performed with antibodies for alpha-actin smooth muscle, uroplakin, cytokeratins and vimentin to assess for the development of detrusor muscle. Embryonic bladder urothelium resulted in ingrowth of fibroblasts into acellular matrix in greater than 85% of 60 grafts consisting of acellular matrix and bladder urothelium. Moreover, there was apparent trans-differentiation of these fibroblasts into a smooth muscle phenotype. This smooth muscle development occurred in a graded, incremental fashion starting as early as 3 weeks and resulting in almost complete repopulation of the matrix at 6 months. These data support the hypothesis that epithelial mesenchymal signaling is important for the differentiation of bladder smooth muscle. Smooth muscle development during augmentation with acellular matrix is facilitated by the placement of epithelia onto the surface of the matrix.