(Verbatim) Currently, two techniques for the induction of bladder regeneration have been preliminarily researched which hold promise for clinical application. The first technology, the in vivo technique, involves the use of a biodegradable scaffold that the host bladder can use to remodel and regenerate a functional bladder. The second technology, the in vitro technique, involves initial establishment of in vitro primary cell cultures from the host's native bladder and has shown promise for use with membranes fabricated from polyglycolic acid polymers (PAP). These cells are then seeded on a biodegradable scaffold to create a composite graft that is then transplanted back into the host for continuation of the regeneration process. The most successful results utilizing in vivo techniques have been with the biomaterial small intestinal submucosa (SIS). In the urinary tract, SIS induces regeneration of bladder that contains all three layers of the bladder (urothelium, smooth muscle and serosa), has contractile activity, and is innervated. Despite these initial encouraging results, there are several concerns that require further investigation prior to the application of this technology for clinical use. These include: 1) Regenerated bladder contains more collagen than normal bladder tissue 2) Limitation in the surface area that can be regenerated with in vivo techniques. The proposed study will address the above two issues with the following specific aims comparing in vivo and in vitro technologies using PAP and SIS membranes: 1) Urinary bladder function in dogs that have undergone in vivo and in vitro bladder reconstruction 2) Neuromuscular function of in vivo and in vitro regenerated smooth muscle using electrophysiological techniques 3) Compliance and viscoelastic properties of in vivo and in vitro regenerated smooth muscle using in vitro stress/strain techniques. 4) Evaluate the morphology of the vascular, connective, and muscular tissue ingrowth that occurs during in vivo and in vitro urinary bladder regeneration. Additionally, evaluate both qualitatively and quantitatively the smooth muscle and extracellular matrix of in vivo and in vitro PAP and SIS-mediated regenerated urinary bladder using standard histological, immunohistochemistry, and molecular techniques. 5) Compare the afferent and automatic efferent fiber innervation of in vivo and in vitro regenerated bladder tissue using immunohistochemistry. Using the canine subtotal cystectomy model, animals will undergo urinary bladder augmentations utilizing unseeded PAP and SIS (in vivo technology) and seeded PAP and SIS (in vitro technology). Animals will have cystometrograms performed preoperatively and at the time of sacrifice: 1,4,8, and 12 months post implantation. Bladder tissue will be harvested and processed for subsequent analysis. The following parameters will be examined: 1) histology, 2) quantitative morphometry, 3) immunohistochemistry for phenotypic markers of cell differentiation and innervation, 4) total DNA, protein, and collagen content, 5) collagen types I and III, and VEGF gene expression by RT-PCR analysis, and 6) in vitro contractility and compliance measurements. Animals with seeded and unseeded SIS will be directly compared to those with seeded and unseeded PAP. If in vivo and/or in vitro tissue engineering techniques prove to be useful clinically, they will revolutionize urologic reconstructive surgery and have wide ranging applications.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-SSS-M (01))
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Mullins, Christopher V
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University of Oklahoma Health Sciences Center
Schools of Medicine
Oklahoma City
United States
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Azzarello, Joseph; Ihnat, Michael A; Kropp, Bradley P et al. (2007) Assessment of angiogenic properties of biomaterials using the chicken embryo chorioallantoic membrane assay. Biomed Mater 2:55-61
Dozmorov, Mikhail G; Kropp, Bradley P; Hurst, Robert E et al. (2007) Differentially expressed gene networks in cultured smooth muscle cells from normal and neuropathic bladder. J Smooth Muscle Res 43:55-72
Zhang, Yuanyuan; Frimberger, Dominic; Cheng, Earl Y et al. (2006) Challenges in a larger bladder replacement with cell-seeded and unseeded small intestinal submucosa grafts in a subtotal cystectomy model. BJU Int 98:1100-5
Kropp, Bradley P; Cheng, Earl Y; Lin, Hsueh-Kung et al. (2004) Reliable and reproducible bladder regeneration using unseeded distal small intestinal submucosa. J Urol 172:1710-3