Preliminary experimental study of urethral reconstruction with tissue engineering and RNA interference techniques

Abstract

This study investigated the feasibility of replacing urinary epithelial cells with oral keratinocytes and transforming growth factor-β1 (TGF-β1) small interfering RNA (siRNA)-transfected fibroblasts seeded on bladder acellular matrix graft (BAMG) in order to reconstruct tissue-engineered urethra. Constructed siRNAs, which expressed plasmids targeting TGF-β1, were transfected into rabbit fibroblasts. The effective siRNA was screened out by RT-PCR and was transfected into rabbit fibroblasts again. Synthesis of type I collagen in culture medium was measured by enzyme-linked immuno sorbent assay (ELISA). Autologous oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts were seeded onto BAMGs to obtain a tissue-engineered mucosa. The tissue-engineered mucosa was assessed morphologically and with the help of scanning electron microscopy. The TGF-β1 siRNA decreased the expression of fibroblasts synthesis type I collagen. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts were seeded onto sterilized BAMG to obtain a tissue-engineered mucosa for urethral reconstruction. The compound graft was assessed using scanning electron microscope. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts had a good compatibility with BAMG. The downregulation of fibroblasts synthesis type I collagen expression by constructed siRNA interfering TGF-β1 provided a potential basis for genetic therapy of urethral scar. Oral keratinocytes and TGF-β1 siRNA-transfected fibroblasts had good compatibility with BAMG and the compound graft could be a new choice for urethral reconstruction.

Figure 1

(a) BAMG Masson staining (×40). (b) BAMG HE staining (×40). (c) Passage 2 oral keratinocytes (×100). (d) Passage 3 fibroblasts (×100). (e) TGF-β1 siRNA-transfected fibroblasts manifested green color fluorescence (×200). (f) Type I collagen concentration curve in transfected cells was significantly lower than the blank control groups. Scale bar=100 μm. BAMG, bladder acellular matrix graft; HE, hematoxylin and eosin; siRNA, small interfering RNA; TGF-β1, transforming growth factor-β1.

Figure 2

(a) Oral keratinocytes seeded onto the BAMG. The cells formed pseudopodia that extended peripherally to allow the cells to connect with each other. Scanning electron microscope (×1000). (b) TGF-β1 siRNA-transfected fibroblasts seeded onto the BAMG. The TGF-β1 siRNA-transfected fibroblasts also adhered to the grafts well. Scanning electron microscope (×1000). Scale bar=10 μm. BAMG, bladder acellular matrix graft; siRNA, small interfering RNA; TGF-β1, transforming growth factor-β1.