Biology Property Of The Porcine Bladder Acellular Matrix And Experiment On Mending Bladder In Canine

PartⅠ: Preparation of porcine bladder acellular matrix and research on its relative biocharacteristics【Objective】To investigate the feasibility of the porcine bladder acellular matrix as biomaterial scaffold for bladder substitution or for tissue engineering application by detecting the physics and biology characteristics and other components.【Methods】The method of hypotension and detergent and nucleic acid enzymatic digestion extraction process has been developed to remove all the cellular components from the porcine bladder to prepare the bladder acelluar matrix. The PBAM was then examined by H-E and Massons staining to confirm no cell elements remained. The methods of the molecular biology and enzyme linked immunosorbent assay (ELISA) were used to determine the contents of the DNA and Transforming Growth Factor-β1 (TGF-β1) respectively in PBAM. The biomechanics of the PBAM were testing by omnipotent electronic material testing machine.【Results】There were no cell elements remained for the PBAM under the examination of optical microscope. The matrix of this scaffold material still have complete three diamensions network structure. The contents of the DNA in PBAM only reduced from 2% to 5% after the initial decellular method. But through the repetition of the double nucleic acid enzymatic digestion and strengthen for the water exchange, the DNA contents were all less than 1%. There were still some growth factor such asTGF-β1 in PBAM. It had stable biomechanics characteristic and had good elasticity and toughness.【Conclusion】The PBAM had good physical rationality and stable biomechanics characteristic. The growth factor for the TGF-β1 in PBAM was first discovered by the method of the ELISA at home and abroad. The discovery of the TGF-β1 in PBAM indicate that the TGF-β1 may have a latent active effects for the construction of the tissue engineering bladder. The PBAM which we made has the character of sterility and asepsis and good biocompatibility and can be stored for long time, so it can be used as ready-made product bioscaffold materials for the tissue engineering and organ substitute or mending.PartⅡ: The immunogenicity and biologic safety in xenotransplantation with porcine bladder aceHular matrix【Objective】To observe the immunogenicity and biologic safety of the bladder acellular matrix graft as biomaterial scaffold for tissue engineering application in exnotransplantation.【Methods】The antigen of Major histocompatibility complex andα-galactosyl residues in the porcine structure cell and endothelial cell were detected by using the method of the immunohistochemistry to observe the effect on this decellularization method. The molecular biology method were used to observe the influence for the PERV in porcine bladder with this decellularization method. The expression of the PERV in canine with bladder substitution for PBAM were still detected.【Results】There are high expressions for the antigens of theα-Gal and MHC-Ⅰin fresh porcine bladder. But the expression of theα-Gal and MHC-Ⅰcut down significantly or nothing after the decellularization course. The gene order of the PERV exist in the fresh porcine bladder genome, but could not found in the PBAM. There were no proof for the alive PERV in exnotransplantation with PBAM.【Conclusion】The major antigen of theα-Gal and MHC-Ⅰdirectly related to the immunological rejection for the xenotransplantation exist in the fresh porcine bladder, but their expression activity cut down significantly or nothing after the decellularization course. There were no expression of the PERV in PBAM. The PBAM have good immunotolerance and reliable biologic safety in xenotransplantation.PartⅢ: Bladder repair and replacement experiment with porcine bladder acellular matrix in vivo for xenogeneic animal canine【Objective】To observe the biocompatibility and biodegradation and biologic safety of the PBAM in different species animal body. We still want to observe the results of the PBAM as the bladder replacement experiment and the influences for bladder regenerate with mending areas. This results can be provided to the next construction the tissue engineering bladder and repair or replacement for the urinary defects.【Methods】hybrid canine were divided into 4 groups and marked with A, B, C and D. Group A with 50% bladder incised and sutured in site directly was control group. As experiment groups, group B, C and D were all incised 50% bladder and replaced using the PBAM respectively (their area was about 30%, 40%, 50% of the overall canine bladder). The blood routine, the electrolytes, the serum creatinine, PERV, cystometry and compliance, plain abdominal radiograph and cystography were detected before and after the operations. One canine in each group will be killed to observe the replacement specimen at 12 weeks and 24 weeks aider the operations.【Results】There were 8 canines living surround the operation stage. The Blood routine and the renal function before and after operations had no differences between the control group and the experimental group. 2 canies in group D died at 28 days and 34 days after the operation. The causes of the death were all induced by vesical perforation, leakage of urine and intra-abdominal infection. After 12 weeks, the vesical capacity regained to 63%, 85%, 89% of their preoperative capacity respectively, and the bladder compliance recovered to 67%, 88% and 87% from their native bladder respectively. Plain abdominal radiograph indicated no bladder stone formation. The bladder specimens couldn't almost find the anastomosis of the scaffold and the native bladder. The scaffold shrinked significantly to 75% to 85%, but new generated bladder tissue with thin wall still could be found. Histology revealed multiplayer urothelial cells and scattered smooth muscle bundle on the replacement bladder. After 24 weeks, the replacement bladder with PBAM regenerated the bladder multiplayer tissue on the whole. After 12 weeks, one canine killed in group B and C could be found the sediment grains cohereing to the replaced bladder with PBAM, but no stone formation in bladder specimens after 24 weeks【Conclusion】As a biomaterial scaffold, PBAM has good biocompatibility and good biodegradable and good biologic safety in xenotransplantation. The initial big animal experiment proved that the PBAM can be used to mend and replace that bladder. But the larger area PBAM still can be induced complications with vesical perforation and scaffold shrink and micro stone formation.