The Experimental Study Of Construction Of Tissue Engineered Corpus Cavernosum And Corpus Spongiosum

CHAPTER ONE THE CULTURE OF SEEDING CELLS1 ObjectiveTo investigate the methods of isolating, culturing and evaluating the rabbit corpus cavernosal smooth muscle cells and endothelial cells.2 MethodsThe explant cell culture and digestion cell culture methods were used to isolate corporal smooth muscle cells from young rabbit cavernosal tissue. Velocity sedimentation were used to enrich the cells. Usingα-smooth muscle actin, myosin and vimentin monoclone mouse antibodies as markers, the cells purity in cultured cells was analysed by immunofluorescence and flow cytometry. The enzymatic digestion methods were used to isolate corpus cavernosal endothelial cells from young rabbit cavernosal tissue. The cells were suspended in endothelial basal medium(EBM-2) supplemented with EGM-2 BulletKit for culturing, Using endothelial specific von Willebrand Factor(vWF) as a marker, the 2nd passage cells was identified by immunofluorescence and transmission electron microscope. Flow cytometry was performed to analyses cells purity.3 ResultsUsingα-smooth muscle actin as the cell marker, the positive rate of corpus cavernosal smooth muscle cells was 16.91% in the digestion cell culture and 11.13% in the explant cell culture. After velocity sedimentation procedures, the cells purity was increased to 26.88% in the digestion cell culture and 21.98% in the explant cell culture. Immunofluorescence confirmed contaminating fibroblasts by vimentin labeling. The corpus cavernosal endothelial cells were cultured in vitro and passaged for six to eight generations. Immunofluorescence confirmed the characteristic of endothelial cells by vWF labeling. Weibel-Palade body (W-P body) was observed by transmission electron microscope. The purity of 2nd passage cells was 93.76%.4 ConclusionIt was difficult to exclude fibroblasts contamination in corporal smooth muscle cells cultured in vitro. Velocity sedimentation procedures could only achieve slight purify of corporal smooth muscle cells. Rabbit corpus cavernosal endothelial cells can be isolated and cultured successfully in vitro by our method.CHAPTER TWO CONSTRUCTION OF CORPUS CAVERNOSUM SMOOTH MUSCLE USING UMBILICAL ARTERY SMOOTH MUSCLE CELLS RESEEDED ON ACELLULAR CORPORAL COLLAGEN MATRICES1 ObjectiveTo investigate the feasibility of constructing tissue engineered corpus cavernosum smooth muscle.2 MethodsAcellular corporal collagen matrices (ACCMs) were obtained from the penis of adult rabbits by a cell removal procedure. ACCMs were implanted into the back muscles of allogenic rabbits to investigate the resulting foreign body reaction. HUASMCs were isolated from human umbilical arteries through explant techniques, and expanded in vitro . Subsequently, third and fifth passage HUASMCs were seeded to ACCMs at a concentration of 30×106 cells/ml. After that, seeded ACCMs were implanted subcutaneously in athymic mice. The implants were retrieved at 10, 20 and 40 days after implantation. Histochemistry, immunohistochemistry and scanning electron microscopy were performed to analyze the morphological characteristics of the engineered tissues. Additionally, organ bath studies were performed to address the contractility of the engineered tissues.3 ResultsThe decellularization process successfully extracted all cellular components and maintained their original collagen fibers. The foreign body reaction to ACCMs consisted of only a transient nonspecific inflammatory response. Light and scanning electron microscopy demonstrated that HUASMCs extended onto the three-dimensional ACCMs scaffolds in vitro. Histologic analyses of the explants from all time points demonstrated a progressive regeneration of smooth muscle, with structures very similar to native corpus cavernosum smooth muscle. The maximum contraction force induced by phenylephrine and electrical stimulation were 3.64±0.18 g/100 mg and 2.50±0.21 g/100 mg.4 ConclusionOur study demonstrates that HUASMCs can be seeded on 3-dimensional ACCM scaffolds and will develop a tissue similar to that of the native corpus cavernosum smooth muscle.CHAPTER THREE FEASIBILITY OF CONSTRUCTING TISSUE-ENGINEERED CORPUS CAVERNOUS URETHRA WITH RABBIT BONE MARROW STROMAL CELLS1 ObjectiveTo investigate the feasibility to construct urethra with PGA and the compatibility of PGA with BMSCs.2 MethodsBMSCs were harvested, and their phenotype was identified. The cells were cultured onto PGA after amplification. BMSCs were labeled with CM-Dil. Histochemistry and scanning electron microscopy were performed to analyze the morphological characteristics of the engineered tissues. The PGA was implanted into the cavernous urethra of six rabbits after partial resection of 1×1 cm full-thickness urethra. Catheter examination and retrograde urethrography were used to evaluate results 1, 2 and 3 months after the operation, and then all dogs were sacrificed for macroscopical and histological examination.3 Results PGA could be seeded with BMSCs in vitro, and BMSCs had the potential of attachment and proliferation on the three-dimensional PGA scaffolds. All dogs survived the procedure.1 of 6 rabbits developed a fistula formation. The other 5 rabbits voided spontaneously without difficulties. Retrograde urethrography revealed no sign of stricture. Histological examination showed the urethral walls were covered by squamous epithelium with slight keratinization.4 ConclusionPGA may be used as the scaffold for BMSCs growing . PGA can results in regeneration of urethral mucosa,but no regeneration of corpus spongiosum.