Engineering the Bladder: Composite Cystoplasty.
Supervisors: Jenny Southgate (Biology, York) and Steve Rimmer (Chemistry,
Sheffield).
For end-stage bladder disease, the most commonly performed surgical procedure to augment or replace the bladder involves incorporating a reconfigured vascularised smooth muscle tissue graft (usually bowel) into the bladder. Although enterocystoplasty can enhance quality of life, it is associated with major complications, including long term risk of cancer. These complications are primarily attributable to the fact that bowel mucosa is structurally and physiologically incompatible with the long-term exposure to urine. To address this, we propose what we have called a “composite cystoplasty” approach, in which the bladder is augmented or reconstructed using a vascularised de-epithelialised smooth muscle host tissue (eg colon) that is lined by autologous urothelium generated in vitro 1,2 .
Our progress to date has included: a) development of robust techniques to propagate normal human urothelial (NHU) cells in vitro 3,4 and identification of the autocrine growth regulatory pathways 5 . b) demonstration that it is possible to induce NHU cells to differentiate to form a tight urothelial barrier 6 and identification of the molecular pathways involved in inducing differentiation 7-10 . c) adaptation of urothelial cell culture techniques for pig, including techniques to produce a differentiated porcine urothelium 11 and development of a surgical model 12 .
In recent work, we have combined in vitro-propagated autologous urothelium with a vascularised de-epithelialised colonic pedicle produced by a technique of extraluminal dissection (Turner, 2010 submitted). The results from this indicate that a differentiated urothelium combined with a host colon-derived smooth muscle successfully forms a functional augmented bladder and effectively overcomes the problems of mucus and calculi seen in conventional enterocystoplasty, without introducing a propensity for fibrosis or contraction.
To take the work forward, we need to improve the efficiency of delivery of urothelium from in vitro to in vivo. This will involve 3 stages: 1) improved harvest of polarised, differentiated urothelial cell sheets from the culture vessel; 2) combination of urothelial sheets with a biodegradable carrier material suitable for surgical transfer and implantation and 3) development of methods to preserve viable urothelial constructs prior to implantation.
1) Improved harvest of the sheet could be provided, while maintaining polarity, by applying a hydrogel sheet functionalised with cell adhesion motifs (e.g. see reference 13). If the hydrogel sheet is functionalised with both cell adhesive peptides and a polymer with good general cell adhesive properties (usually achieved by balancing the hydrophobic-hydrophilic balance and surface porosity 14 ) cells can be lifted from less well-performing substrates.
15 In sheet format it might also be possible to include the developing extra-cellular matrix with the lifted cells.
2) Several newer biodegradable polymers, which could provide much improved control of degradation with improved cell adhesion, are available. In particular polysaccharide-synthetic hydrogels would be attractive. Such materials degrade to sugars and synthetic oligomers both of which are less toxic than lactic and glycolic acids. Cells generally show poor adhesion to many of the

available supports (eg Poly(lactic acid-co-glycolic) acid) but cost –effective improvements are possible using either amine functionalisation and/or controlling the hydrophobic-hydrophilic balance.
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References
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Bolland F, Southgate J. Bio-engineering urothelial cells for bladder tissue transplant.
Expert Opin Biol Ther. 2008 Aug;8(8):1039-49.
Turner AM, Southgate J. The tissue-engineered urinary bladder. In: Meyer U, Meyer
T, Handschel J, Wiesmann HP (eds). Fundamentals of tissue engineering and regenerative medicine: Springer, 2009.
Southgate J, Hutton KA, Thomas DF, Trejdosiewicz LK. Normal human urothelial cells in vitro: proliferation and induction of stratification. Laboratory Investigation.
1994;71:583-94.
Southgate J, Masters JR, Trejdosiewicz LK. Culture of Human Urothelium. In:
Freshney RI, Freshney MG (eds). Culture of Epithelial Cells. 2nd edition ed. New
York: J Wiley and Sons, Inc., 2002:381-400.
Varley C, Hill G, Pellegrin S, et al. Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro. Exp Cell Res. 2005
May 15;306(1):216-29.
Cross WR, Eardley I, Leese HJ, Southgate J. A biomimetic tissue from cultured normal human urothelial cells: analysis of physiological function. Am J Physiol Renal
Physiol. 2005 Aug;289(2):F459-68.
Varley CL, Bacon EJ, Holder JC, Southgate J. FOXA1 and IRF-1 intermediary transcriptional regulators of PPARgamma-induced urothelial cytodifferentiation. Cell
Death Differ. 2009 Jan;16(1):103-14.
Varley CL, Garthwaite MA, Cross W, Hinley J, Trejdosiewicz LK, Southgate J.
PPARgamma-regulated tight junction development during human urothelial cytodifferentiation. J Cell Physiol. 2006 Aug;208(2):407-17.
Varley CL, Stahlschmidt J, Lee WC, et al. Role of PPAR {gamma} and EGFR signalling in the urothelial terminal differentiation programme. J Cell Sci. 2004 Apr
15;117(Pt 10):2029-36.
Varley CL, Stahlschmidt J, Smith B, Stower M, Southgate J. Activation of Peroxisome
Proliferator-Activated Receptor-gamma Reverses Squamous Metaplasia and Induces
Transitional Differentiation in Normal Human Urothelial Cells. Am J Pathol. 2004
May;164(5):1789-98.
Turner AM, Subramaniam R, Thomas DF, Southgate J. Generation of a functional, differentiated porcine urothelial tissue in vitro. Eur Urol. 2008 Dec;54(6):1423-32.
Fraser M, Thomas DF, Pitt E, Harnden P, Trejdosiewicz LK, Southgate J. A surgical model of composite cystoplasty with cultured urothelial cells: a controlled study of gross outcome and urothelial phenotype. BJU Int. 2004 Mar;93(4):609-16.
“Cell adhesive hydrogels synthesized by copolymerization of arg-protected Gly-Arg-
Gly-Asp-Ser methacrylate mo nomers and enzymatic deprotection” L. Perlin, S.
MacNeil, S. Rimmer, Chem. Commun., 5951 (2008)
“Culture of dermal fibroblasts and protein adsorption on block conetworks of poly(butyl methacrylate-block-(2,3 propandiol-1-methacrylate-stat-ethandiol dim ethacrylate))” Y. Sun , J. Collett , N.J. Fullwood, S. Mac Neil, S. Rimmer,
Biomaterials , 28 661 (2007)
”Sub-micron poly(N-isopropyl acrylamide) particles as temperature responsive vehicles for the detachment and delivery of human cells” S. Hopkins, S.R. Carter,
J.W. Haycock, N.J. Fullwood, S. MacNeil, S. Rimmer, Soft Matter , 5 4928 (2009)
“Cytocompatibility of poly(1,2 propandiol methacrylate) copolymer hydrogels and conetworks with or without alkyl amine functionality” S. Rimmer, Stacy-Paul Wilshaw,
P. Pickavance, E. Ingham, Biomaterials , 30 2468 (2009)