Urethral Reconstruction Using Bone Marrow Mesenchymal Stem Cells And Smooth Muscle Celle Seeded Bladder Acellular Matrix

Backgrounds and objectives:Congenital or acquired abnormalities may lead to urethral injure or loss, often needing surgical reconstruction. With the popularity of the endoscopic surgery during the past decades, urethral damage have become more common. Although surgical methods of urethroplasty have been developed rapidly, these methods often lead to many complications, including urethrocutaneous fistula and strictures. Especially complex urethral stricture remains one of the most difficult problems to be solved in urology. The reason of urethral stricture is the lack of postoperative protection of urethral mucosal, leading to scarring. So it is one of the key factor of successful urethral operation to promote urethral mucosa growth. Various tissues, such as bladder mucosa, genital and extragenital skin flaps, colonic mucosa lingual mucosa and buccal mucosa have been used for experimental urethroplasty. Nonetheless, many complications also have been reported. More importantly, these tissues are obtained limitedly when treating long urethral stricture. Tissue-engineered urethral graft could potentially serve as an selective source of graft for urethral reconstruction. In this study, we isolated and expanded BMSCs and smooth muscle cells (SMCs) in vitro and seeded into the two side of BAM graft to construct tissue-engineered sheet graft (TESG). Then the TESG was transplanted into the omentum of rabbit for2weeks and applied to urethral reconstruction in rabbit.Methods:BMSCs and SMCs of rabbit were isolated、expanded、and identified in vitro. Then BMSCs were inoculated onto the BAM of3.0×1.5cm2at a density of1×106/cm2and cultured for3days. Then SMCs were inoculated onto the other side of BAM at a density of1x106/cm2and cultured for3days. The TESG was constructed and transplanted into the omentum of rabbit for2weeks before urethral reconstitution. Meanwhile, BAM was transplanted into the omentum as the control group. BAM only was as the control group. Then the graft was used to construct TESG and implanted into the omentum of rabbit for2weeks before urethral reconstruction. In experimental group,24male rabbits, each2.0-2.5kg, divided into four groups and each of six, were given to fasting8hours before surgery. Another six rabbits were as the control group, rabbits were anesthetized with intravenous injection of30mg/kg pentobarbital sodium preoperatively and the8Fr silicone catheters were inserted into the urethra of rabbits. In the experimental group, A ventral urethral defect of3cm in length and1.5cm in diameter was made and the urethral defect was about2.0cm from the external urethral orifice. The TESG was sutured on the urethral defect using5-0absorbable sutures. In the control group, unseeded BAM was sutured on the urethral defect. The8Fr silicone catheter was maintained until2week postoperatively. The specimens of TESG were respectively harvested at2weeks,4weeks,8weeks and16weeks after operation and stained with hematoxylin and eosin. Immunohistochemical analysis was performed using primary antibody to CK AE1/AE3、 uroplakin Ⅲa and a-SMA to detect the regeneration of the epithelium and smooth muscle. Retrograde urethrography was performed at16weeks postoperatively.Results:Three days after culture, the adherent BMSCs were scattered on the culture flask bottom and the nonadherent cells were washed off. Seven days after culture, the BMSCs proliferated rapidly and merged into lines. The adherent cells had the characteristic Structure of fusiform morphology. SMCs had the typical morphology of spindle-shaped. After the decellularization processing, the BAM was a porous membrane. Hematoxylin and eosin staining of BAM revealed no residual cells. Scanning electron microscopy also revealed no residual cells adhering to the grafts. After2weeks of omental maturation, thin and mature epithelial layer was formed. All rabbits in the experimental group survived until sacrifice. The operative incision completely healed within2week postoperatively. After catheter removal, all rabbits had no dysuresia. At8weeks, there was no difference between the graft and the normal urethra and no severe shrinkage was observed. By2weeks, HE stain showed that the TESG was completed covered with thin epithelium. The interconnecting areas between the TESG and native urethra could be easily identifed, because the urothelium of native urethra was thicker than that of TESG. By4weeks after TESG grafting in vivo, the urothelium covering the TESG became thicker and organized smooth muscle bundles were observed. At8weeks and16weeks after TESG grafting in vivo, multilayer urothelium covered the graft.. Immunohistochemisty showed the epithelial layer was stained positively for CK AE1/AE3and uroplakin Ⅲa. With the time going on, epithelial layer became more and more thicker, which was proven by the HE staining and immunohistochemical staining. Organized smooth muscle bundles were also observed. There were neovascularization within the center of TETG. Retrograde urethrography demonstrated diverticula formation or no urethral stricture. Three rabbits in control group were dead within4weeks postoperatively. Autopsy showed urethras were almost completely blocking. Another three rabbits obvious urethral stricture. The reason for the stricture or blocking of the urethra in control group was that tissue-engineered urethra in control group was short of the protection of the urothelium, causing urine extravasation and scar formation.Conclusions:we successfully constructed a TESG using BMSCs and SMCs seeded BAM for urethral reconstruction. The results have showed that BMSCs can be potentially promoted urothelial regeneration for tissue engineered urethra.