Tissue-engineered tubular substitutions for urinary diversion in a rabbit model

Abstract

Clinically, autologous gastrointestinal segments are traditionally used for urinary diversion. However, this procedure often causes many serious complications. Tissue engineering may provide an alternative treatment method in urinary diversion. This research aims to produce tissue-engineered tubular substitutions by using homologous adipose-derived stem cells, smooth muscle cells, and bladder acellular matrix in developing urinary diversion in a rabbit model. Adipose-derived stem cells and smooth muscle cells of rabbit were obtained and cultured in vitro. These cultured adipose-derived stem cells and smooth muscle cells were seeded onto the two sides of the bladder acellular matrix and then incubated for seven days. The cell-seeded matrix was used to build tissue-engineered tubular substitutions, which were then implanted and wrapped into the omentum in vivo for two weeks to promote angiogenesis. In the experimental group, the bladder of 20 rabbits was totally resected, and the above tissue-engineered tubular substitutions were used for urinary diversion. In the control group, bladder acellular matrix tubular substitutions with unseeded cells were implanted into the omentum and were used as urinary diversion on another five rabbits with the same process. The implants were harvested, and histological examination was conducted at 2, 4, 8, and 16 weeks after operation. Intravenous urography assessment was performed at 16 weeks postoperatively. All the rabbits were alive in the experimental group until they were sacrificed. Histological analysis of the construct displayed the presence of multilayer urothelial cells on the luminal side and organized smooth muscle tissue on the other side, and different diameters of neovascularization were clearly identified in the substitutions obtained. No leakage, stricture, or obstructions were noted with intravenous urography assessment. All the animals in the control group died within two weeks, and urine leakage, scar formation, and inflammation were detected through autopsy. This study demonstrates the feasibility of tissue-engineered tubular substitutions constructed using homologous adipose-derived stem cells, smooth muscle cells, and bladder acellular matrix for urinary diversion in a rabbit model.

Keywords: Tissue engineering; adipose-derived stem cells; bladder acellular matrix; epithelium; omentum; smooth muscle cells; urinary diversion.

Figure 1

Morphological characterization and identification of ADSCs. (a) Primary culture after three days (× 100). (b) After incubation for 10 days, the cells proliferated rapidly and displayed a spindle fibroblastic appearance (×100). Flow cytometry analysis demonstrated expression of (c) CD90 (99.98%) and (d) CD44 (99.94%), and there was no significant expression of (e) CD34 (2.73%), and (f) CD45 (2.55%). (A color version of this figure is available in the online journal.)

Figure 2

Morphological characterization and immunofluorescence staining of SMCs. (a) After three days of primary culture, the cells proliferated on the surfaces of Petri dish and reached about 30% confluence (×100). (b) After seven days of culture, the cells reached 80%–90% confluence and displayed a classic spindle-shaped morphology (×100). (c) α-SMA immunofluorescence staining (in green) of SMCs in combination with DAPI cell nuclei stain (in blue) (× 200). (A color version of this figure is available in the online journal.)

Figure 3

Characteristics of BAM. (a) After decellularization procedure, the BAM appeared as a translucent film. (b) H&E staining of BAM (×400). (c) Masson’s trichrome staining of BAM (×400). (d) Sirius Red staining of BAM (×400). (e) Scanning electron microscope of BAM (×400). (A color version of this figure is available in the online journal.)

Figure 4

The feature of BAM seeding. After seeding cells onto the BAM, the cells fused well on the three-dimensional BAM scaffold. (a) H&E staining of luminal side (×400). (b) H&E staining of outside side (×400). (c) Scanning electron microscope of luminal surface (×2000). (d) Scanning electron microscope of outside surface (×2000). (A color version of this figure is available in the online journal.)

Figure 5

Regeneration of epithelium and neovascularization after omental incubation. (a) H&E staining of TETSs (× 400). (b) H&E staining of the unseeded BAM (× 400). (c) Anti-AE1/AE3 immunohistochemistry staining displayed a one-layer epithelium structure (× 200). (d) Anti-AE1/AE3 immunohistochemistry staining showed no obvious epithelium in the unseeded BAM (× 200). (e) Anti-CD31 immunohistochemistry staining revealed neovascularization of TETSs (× 200). (f) Anti-α-SMA immunohistochemistry staining revealed vascular walls in TETSs (× 200). (A color version of this figure is available in the online journal.)

Figure 6

Histologic characteristics of TETSs in the experimental group after urinary diversion at 2, 4, 8, and 16 weeks. (a, b, c, and d) H&E staining displayed the regeneration of epithelium layers of TETSs (× 400). (e–h, i–l, and m–p) Immunohistochemical staining of AE1/AE3, uroplakin IIIa, and ZO-1 revealed the regeneration of epithelium of TETSs, respectively (× 400). (A color version of this figure is available in the online journal.)

Figure 7

Immunohistochemistry analysis for regeneration of smooth muscle and neovascularization at 16 weeks (× 200). (a) Anti-α-SMA antibody positive displayed organized smooth muscle tissues. (b)Anti-CD31 antibody positive showed well-developed angiogenesis. (c) Anti-α-SMA antibody positive revealed vessel wall. (A color version of this figure is available in the online journal.)

Figure 8

Intravenous urography observation. Intravenous urography at 16 weeks postoperatively showed urinary tract was unblocked and that no leakage, stricture, or obstruction occurred in the TETSs and bilateral ureters. (A color version of this figure is available in the online journal.)