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Journal of Pediatric Urology (2015) xx, 1.e1e1.e6

aUrology Department,University of California, Irvine,Orange, CA, USA

bPathology Department,University of California, Irvine,Orange, CA, USA

cHospital for Sick Kids, Toronto,ON, Canada

Correspondence to:A. E. Khoury, Department ofUrology, University ofCalifornia, Irvine, 505 S. MainStreet, Suite 100, Orange, CA92868, USA

aekhoury@uci.edu(A.E. Khoury)

Keywords

Aerosol; Bladder augmentation;Demucosalized colonic segment

Received 9 June 2014Accepted 26 February 2015Available online xxx

Please cite this article in precolonic segments for bladderdx.doi.org/10.1016/j.jpurol.2

http://dx.doi.org/10.1016/j.j1477-5131/ª 2015 Journal of P

Aerosol transfer of bladder urothelialand smooth muscle cells ontodemucosalized colonic segments forbladder augmentation: In vivo, longterm, and functional pilot study

Guy Hidas a, Hak J. Lee a, Andrej Bahoric c, Maryellen S. Kelly a,Blake Watts a, Zhongbo Liu a, Samah Saharti b, Achim Lusch a,Alireza Alamsahebpour a, David Kerbl a, Hung Truong a, Xiaolin Zi a,Antoine E. Khoury a

Summary

BackgroundBladder augmentation technique has changed over theyears and the current practice has significant adversehealth effects and long-term sequelae. Previously, we re-ported a novel cell transfer technology for covering demu-cosalized colonic segments with bladder urothelium andsmooth muscle cells through an aerosol spraying of thesecells and a fibrin glue mixture.

ObjectiveTo determine the long-term durability and functionalcharacteristics of demucosalized segments of colon repo-pulated with urothelial cells in the bladder of swine for usein augmentation cystoplasty.

Study designNine swine were divided into three groups. The first group(control) underwent standard colocystoplasty; the secondgroup underwent colocystoplasty with colonic demucosali-zation and aerosol application of fibrin glue and urothelialcell mixture; in the third group detrusor cells were added tothe mixture described in group two. The animals were keptfor 6 months. Absorptive and secretory function wasassessed. Bladders were harvested for histological andimmunohistochemical evaluation.

ResultsAll animals but one in the experimental groups showedconfluent urothelial coverage of the colonic segment in the

Figure One animal in group three showed an extand massive collagen deposition.

ss as: Hidas G, et al., Aerosol transfer of bladder urotaugmentation: In vivo, long term, and functional pilot015.02.020

purol.2015.02.020ediatric Urology Company. Published by Elsevier Ltd. A

bladder without any evidence of fibrosis, inflammation, orregrowth of colonic epithelial cells. Ten percent of theinstilled water in the bladder was absorbed within an hourin the control group, but none in experimentalgroups(p Z 0.02). The total urine sediment and proteincontents were higher in the control group compared withexperimental groups (p < 0.05).

DiscussionBoth study groups developed a uniform urothelial lining.Histologically, the group with smooth muscle had an addedlayer of submucosal smooth muscle. Six months afterbladder augmentation the new lining was durable. We werealso able to demonstrate that the reconstituted augmentedsegments secrete and absorb significantly less than thecontrol colocystoplasty group. We used a non-validatedsimple method to evaluate permeability of the new uro-thelial lining to water. To determine if the aerosol transferof bladder cells would have behaved differently in theneurogenic bladder population, this experiment shouldhave been performed in animals with neuropathicbladders.

ConclusionAerosol spraying of single cell suspension of urothelial andmuscular cells with fibrin glue resulted in coverage of thedemucosalized intestinal segment with a uniform urotheliallayer. This new lining segment was durable withoutregrowth of colonic mucosa after 6 months. The newreconstituted segment absorbs and secretes significantlyless than control colocystoplasty.

ensive scar with sloughed urothelium, fibroblasts,

helial and smooth muscle cells onto demucosalizedstudy, Journal of Pediatric Urology (2015), http://

ll rights reserved.

1.e2 G. Hidas et al.

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Introduction

Congenital and acquired defects, such as bladder exs-trophy, myelomeningocele, spinal cord lesions, and bladderoutlet obstruction, are associated with poor bladder stor-age caused by decreased capacity, abnormal contractility,and poor compliance. This can result in incontinence,infection, vesicoureteric reflux, and renal injury. Recon-struction of this type of bladder using intestinal segmentsprevents these complications and today is considered thestandard of care for managing these end-stage bladders.However, isolation of these segments from the gastroin-testinal tract and incorporating them into the urinary tracthas been associated with significant long-term sequelaebecause of the existing secretory and absorptive functionsof the intestinal mucosa [1,2]. The secretion of mucus inthe urine is associated with infection and urinary stoneformation. Exposure of the absorptive surface of the in-testinal mucosa to urine leads to metabolic derangementsand electrolyte abnormalities. Another rare complication ismalignant transformation of the intestinal mucosa, a riskthat requires lifelong endoscopic follow-up of these pa-tients [3].

Several natural and synthetic materials have beenevaluated for bladder augmentation to avoid the drawbacksof intestinal bladder augmentation. However, ideal mate-rials for bladder augmentation are not yet available. Theintroduction of tissue engineering technology for bladdersubstitution has been considered a promising alternativeapproach [4,5]. However, these tissue-engineered bladdershave behaved like grafts, with inadequate delivery of ox-ygen and nutrients to the transplanted cells and the in-efficiency of waste removal. As a result of poor graft take,scarring of these tissue-engineered bladders was frequentlyobserved without improvement in bladder capacity [6]. Useof demucosalized intestinal segments to overcome theabsorptive and secretory complications is often associatedwith shrinkage of the segment and regrowth of thegastrointestinal epithelium [7,8].

To solve the issues of shrinkage and regrowth ofgastrointestinal epithelium in demucosalized segments,we have substituted the intestinal mucosa with autolo-gous bladder urothelium and muscle. We have reportedpreviously a novel cell transfer technology for coveringdemucosalized colonic segments with bladder urotheliumand smooth muscle cells through aerosol spraying of thesecells and a fibrin glue mixture [9,10]. These segmentswere used for immediate bladder augmentation. After apilot study for the feasibility of this novel technology [9],a 6-week controlled experiment demonstrated that thecolonic segments used for bladder augmentation hadconfluent epithelial coverage without fibrosis andregrowth of intestinal mucosa [10]. However, the long-term durability of this lining and the absorptive andsecretory function of the resurfaced segment remainedunknown.

Therefore, in this study, we aimed to determine whetherthis new urothelial lining is impermeable, non- or lesssecretory, and will persist over a long period of time(6 months) without regrowth of colonic epithelium.

Please cite this article in press as: Hidas G, et al., Aerosol transfer ocolonic segments for bladder augmentation: In vivo, long term, and fdx.doi.org/10.1016/j.jpurol.2015.02.020

Subjects and methods

Experimental design

The study included nine Yucatan pigs weighing 20 kg each.The animals were divided into three equal groups and un-derwent one of the following procedures: group 1 (controlgroup), classic colocystoplasty; group 2, demucosalizedcolocystoplasty with aerosol application of fibrin glue andsingle cell suspension of urothelial cells; group 3, demu-cosalized colocystoplasty with aerosol application of fibringlue and single cell suspension of urothelial and smoothmuscle cells. The animals were kept for 6 months, afterwhich they underwent endpoint surgery.

Methods and procedures

Preoperative preparation and anesthetic considerations:The experimental protocol was reviewed and approved bythe university animal research committee. Animal handlingand all procedures were conducted following the institu-tional animal care and use committee guidelines and withthe university’s veterinary supervision.

Animal bowel preparation: The animals were fed Ensure(Abbott Laboratories, IL, USA) for 1 day, transitioned to aclear liquid diet for another 1 day, and then fasted over-night before the surgery.

Anesthesia and prophylactic antibiotics: After intra-muscular injection of telazol and xylazine, the animalswere intubated and maintained on isoflurane anesthesiathroughout the surgery. Prophylactic metronidazole(10 mg/kg) was given via intravenous injection during sur-gery. In addition, enrofloxacin was given intramuscularlyjust before surgery and every 12 hours as a prophylacticantibiotic for 48 hours thereafter.

Surgical procedure

In all groups: The peritoneal cavity was entered through amidline abdominal incision followed by removal of a2 � 2-cm segment of bladder for preparation of urothelialand smooth muscle suspension (see Urothelial cellpreparation).

Group 1: A 10-cm segment of the sigmoid colon wasmobilized and then mesenteric exclusion was performed ofthe blood supply of the sigmoid segment. Continuity of thecolon was restored using interrupted 3-0 polyglycate su-tures. The isolated segment was opened along the anti-mesenteric border and reconfigured into a U-shaped patch,which was used for bladder augmentation. The colonicsegment was anastomosed to the bladder with a singlelayer of continuous 3-0 polyglycate suture.

Groups 2 and 3: A 16F Foley catheter was insertedthrough the anus and the balloon of the Foley catheter wasmilked into the middle of the sigmoid colon. A 10-cmsegment of the sigmoid colon was mobilized and the lumenwas occluded using two vessel loops brought through holesin the sigmoid mesentery. The isolated sigmoid segmentwas filled with sterile water through the Foley catheter to

f bladder urothelial and smooth muscle cells onto demucosalizedunctional pilot study, Journal of Pediatric Urology (2015), http://

Figure 1 Hematoxylin and eosin-stained sections of thereconstructed segment of urothelial cells (group 2) shownormal appearing, multilayered urothelial-like epithelium withno evidence of colonic mucosa regrowth or suburothelialfibrosis. 20� magnification.

Aerosol transfer of bladder urothelial 1.e3

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facilitate colonic dissection of the seromuscular layer offthe mucosal layer of the sigmoid as described by Lima [11].The seromuscular layer was incised along the anti-mesenteric border followed by elevation of this layer offthe mucosa along the entire circumference from both sides.The 10-cm isolated seromuscular segment was transferredmedially with its mesentery. The redundant mucosa wasreplaced into the lumen and the incised edges of the colonwere reapproximated using interrupted 3-0 polyglycatesutures.

In group 2, the isolated demucosalized segment wassprayed with fibrin glue and urothelial cells, while in group3, the surface was sprayed with fibrin glue and a mixture ofurothelial cells and smooth muscle cells. The bladder wasopened in a clam fashion and the constructs were then usedfor bladder augmentation with a single layer of continuous3-0 polyglycate sutures between the bladder and thecolonic segment. The abdominal incision and skin wereclosed. We did not use any catheters or drains in thepostoperative period based on the experience gained in ourprevious short-term experiments [9,10]. These studiesconfirmed that it was safe to perform the procedurewithout using catheters as these animals had normal blad-ders without storage or voiding issues.

Pain was managed with buprenorphine every 8 hours forthe first 48 hours and then buprenorphine was given asneeded. Postoperative care also included administration ofantibiotics for 48 hours. Feeding resumed slowly until theanimal initiated a bowel movement.

Urothelial cell preparation

Urothelial cell preparation was performed, as previouslydescribed [9] simultaneously with the colonic demucosali-zation procedure. A full thickness 2 � 2-cm segment ofbladder was harvested. The urothelial lining was separatedfrom the remainder of the segment and each was placed ina separate tube. The tissue samples were minced intosmaller pieces and then digested with 10 mg of collagenaseIV in 5 ml of keratinocyte serum-free media for 1.5 hours.The resultant urothelial and muscle single cell suspensionswere individually centrifuged at 600 g for 3 minutes. Themedium was discarded, and 5 ml of autologous porcineserum was added to the cell suspension.

The cell suspension was aerosolized and sprayed on tothe demucosalized sigmoid segment concomitantly with5 ml of fibrin glue using a tri-nozzle air compressor sprayerwith separate compartments for the cells and the fibringlue. The mixture was left to adhere on the demucosalizedsurface for 10 minutes.

Endpoint surgery

Secretion test: After administration of anesthesia, 50 ml ofurine was collected and centrifuged. The solid precipitatewas weighed and expressed as mg/ml of urine. The totalprotein content in the urine for the different animals wasmeasured using the Lowry protein assay kit following theinstructions provided by the manufacturer.

Absorption test: After ligation and division of both ure-ters, the bladder was drained and then filled with 300 ml of

Please cite this article in press as: Hidas G, et al., Aerosol transfer ocolonic segments for bladder augmentation: In vivo, long term, and fdx.doi.org/10.1016/j.jpurol.2015.02.020

distilled water. The residual volume of distilled water in thebladder was measured after 1 hour to evaluate theabsorptive capacity of the augmented segment.

Histological studies: The augmented segments wereharvested and fixed in 10% buffered formalin for 48 hoursfollowed by embedding in paraffin. Serial sections of theconstruct, representing peripheral and central areas ofeach segment, were stained with hematoxylin and eosinand examined by a single pathologist. Masson trichromewas used to stain for muscle and collagen, and periodic acidSchiff (PAS) was used to detect polysaccharides in mucosalcells. Immunohistological staining with mouse monoclonalanti-cytokeratin peptide 7 (clone LDS-68) and rabbit poly-clonal anti-uroplakin III was performed in groups 2 and 3 toreveal terminal urothelial differentiation of the sprayedcolonic segments.

Results

Histological studies

Hematoxylin and eosin stained sections showed evidence ofnormal colon mucosa in group 1. All of the animals in groups2 and 3 did not show any evidence of colonic mucosaregrowth (Figs. 1 and 2, respectively). All animals in group2, and all but one in group 3, showed normal appearingmultilayered urothelial-like epithelium with a spontane-ously segregated underlying layer of bladder smooth musclecells resting on colonic submucosa with minimal fibrosis, aswell as evidence of underlying neo-vascularization. Acontinuous colonic submucosal layer was seen separatingthe colonic muscle layers from the newly formed detrusormuscle layer. PAS staining of the constructs showed noevidence of colonic mucosa regrowth, there was minimalfibrosis with mild inflammatory cell infiltrates in Massontrichrome-stained sections. One animal in group 3 showedan extensive scar with sloughed urothelium, fibroblasts,and massive collagen deposition (Fig. 2).

f bladder urothelial and smooth muscle cells onto demucosalizedunctional pilot study, Journal of Pediatric Urology (2015), http://

Figure 2 Hematoxylin and eosin-stained sections of thereconstructed segment of both urothelial and smooth musclecells (group 3) show normal appearing, multilayered urothelial-like epithelium with no evidence of colonic mucosa regrowth orsuburothelial fibrosis. Light microscopy, 2� magnification,inset 20� magnification.

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Immunohistochemistry

To demonstrate that the newly formed hybrid constructedepithelium was indeed true urothelium, immunohisto-chemical analyses for various urothelium-specific differ-entiation markers were performed. There was a uniformpositive staining for cytokeratin 7 (Fig. 3) in mostepithelial cells of reconstructed segments. Monoclonalantibodies against uroplakin III, a transmembrane protein,which constitutes a specific terminal differentiationproduct of urothelial cells showed a strong continuouslinear outline of the superficial cell membrane of theumbrella cells.

Absorptive studies

There was no reduction in the volume of instilled water inthe experimental groups after 1 hour of observation,

Figure 3 (A) Cytokeratin 7 expression is uniformly positive in areconstructed segment with sprayed urothelial and smooth musclemagnification. (B) Uroplakin III expression by superficial umbrellareconstructed segment with sprayed urothelial and smooth musclemagnification.

Please cite this article in press as: Hidas G, et al., Aerosol transfer ocolonic segments for bladder augmentation: In vivo, long term, and fdx.doi.org/10.1016/j.jpurol.2015.02.020

compared with the control group in which an average of10% of the instilled water was absorbed (273 � 6 ml)(p Z 0.027).

Sediment and protein content

Total urine sediments and total protein contents weresignificantly lower in groups 2 and 3 when compared withthose in the control group (Table 1). The control grouphad mean total urine sediment of 3.52 mg/ml (SD � 0.38).Group 2 had mean total urine sediment of 1.52 mg/ml(SD � 0.74) and group 3 had a mean of 1.39 mg/ml(SD � 0.017). Statistical significance (p < 0.01) was foundfor both groups 2 and 3 when compared with the totalsediment level in the control group. Urine protein levelswere also significantly different (p < 0.01) between thecontrol group and groups 2 and 3. The control group had amean urine protein concentration of 0.82 mg/ml(SD � 0.13), group 2 had a mean of 0.02 mg/ml(SD � 0.02), and group 3 had a mean of 0.08 mg/ml(SD � 0.05).

Discussion

This study demonstrates the long-term durability of bladderaugmentation by a novel technology using demucosalizedsigmoid colon that was repopulated with bladder urothelialand smooth muscle cells. These cells were prepared fromthe animal’s own bladder tissue and mixed with fibrin gluefor spraying.

Previously, Hafez et al. showed that 4 weeks afteraugmentation with aerosol sprayed demucosalized colon, ahistological intact urothelial layer is apparent with arandomly aligned, but distinctly segregated layer of smoothmuscle cells [9]. This concept was further tested in a 6-week controlled study, which showed that demucosalizedcolocystoplasty without the cellular component of thespray was associated with severe fibrosis and contracture;however, when the demucosalized segment was repopu-lated using the aerosol spraying technology with eitherbladder urothelial cells or a mixture of bladder urothelial

ll layers of neo-urothelium of demucosalized colocystoplastycells (group 3). Immunohistochemistry, light microscopy, 20�cell layer of neo-urothelium of demucosalized colocystoplastycells (group 3). Immunohistochemistry, light microscopy, 20�

f bladder urothelial and smooth muscle cells onto demucosalizedunctional pilot study, Journal of Pediatric Urology (2015), http://

Table 1 Urine total sediment and protein concentration in the different study groups.

Group Urine total sediment, mg/ml (mean � SD) Urine protein, mg/ml (mean � SD)

Control 3.52 � 0.38 0.82 � 0.13Urothelial only 1.52 � 0.74a 0.02 � 0.02a

Urothelial and muscle 1.39 � 0.017a 0.08 � 0.05a

a p < 0.01 when compared with the control group.

Aerosol transfer of bladder urothelial 1.e5

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and smooth muscle cells, a layer of neo-urothelium thatrested directly on the colonic submucosa was generatedwithout fibrosis or shrinkage [10]. Furthermore, the mixedurothelial and smooth muscle cells in the suspensiondemonstrated a striking ability to organize spontaneouslyinto a luminal epithelial and subepithelial muscle layer[10].

The rationale for the using both urothelial and smoothmuscle cell suspension came from Baskin’s previous work[12], which showed that mesenchymal-epithelial in-teractions are necessary for the development of thebladder smooth muscle, and the mechanism responsiblefor this interaction involves locally diffusible growthfactors. In our study we found that both groups developeda robust and uniform urothelial lining. Histologically, thegroup with smooth muscle had an added layer of sub-mucosal smooth muscle. The addition of the detrusorsmooth muscle did not make the experimental proceduremore complex; however, we are unable to demonstrateany objective benefit from this additional layer in ourstudy.

We used a non-validated simple method to evaluatepermeability of the new urothelial lining to water. Byoccluding both ureters, we ensured that no additionalvolume entered the bladder and so the change in the vol-ume of instilled water could be used as a surrogate forabsorption. We had performed an extensive literaturesearch and failed to find a validated method to evaluatethe absorption ability of augmented bladders in pigs. Moreresearch is needed to develop a precise and validatedmethod.

Before translating this technology to clinical applicationin human subjects there were additional questions thatneeded to be answered. What is the long-term durability ofthis lining? Will these newly reconstructed urothelial cellscontinue to multiply and renew? Or will intestinal mucosalcells regrow and repopulate the original segment?Furthermore, functional properties of the augmentedsegment needed to be explored. More specifically, is thenew urothelial lining impermeable? Lastly, will this tech-nique effectively prevent mucus secretion by the recon-structed augmented segment? Our long-term study wasdesigned to try to answer these questions. We showed that6 months after bladder augmentation the new lining wasdurable. There was no evidence of colonic epithelialregrowth, the urothelial cells continued to renew and toform a consistent uniform urothelial layer. We were alsoable to demonstrate that the reconstituted augmentedsegments secrete and absorb significantly less than thecontrol colocystoplasty group.

We had one failed procedure in which the sprayed cellsdid not repopulate the demucosalized segment and an

Please cite this article in press as: Hidas G, et al., Aerosol transfer ocolonic segments for bladder augmentation: In vivo, long term, and fdx.doi.org/10.1016/j.jpurol.2015.02.020

extensive inflammatory and fibrotic reaction was seen. Webelieve that this severe fibrosis might be attributed to thefailure of the urothelial cells to seed on the demucosalizedbed, therefore resulting in exposure of the demucosalizedsegment to urine. We do not have a definitive explanationfor this one failed experiment.

To determine if the aerosol transfer of bladder cellswould have behaved differently in the neurogenic bladderpopulation, this experiment should have been performed inanimals with neuropathic bladders. However, long-termmaintenance of an animal model with neurogenicdysfunction is very challenging. Although we do not antic-ipate that the dissociated urothelium and smooth musclecells on a colonic matrix would behave differently, thisquestion will obviously remain unanswered until such timeas this technology is ready to be applied and tested in a fewpilot human cases.

One of the advantages of this technology is the ability toobtain a cell suspension in a single operative settingwithout the need for ex vivo cell culture and expansion. Inthis study, the bladder dissociation was processed in oururology basic science laboratory. As the ultimate goal ofthis technology is to translate this work to pediatric andadult patients who require bladder augmentation or sub-stitution, we are currently working on a method to performthe cellular dissociation by the surgical team in the oper-ation room. We hope to be able to simplify the procedureand make it widely available for every standard operatingroom without the need for sophisticated tissue culturelaboratory facilities.

Conclusion

Our long-term animal study demonstrated that aerosolspraying of single cell suspension of urothelial and muscularcells with fibrin glue resulted in coverage of the demuco-salized intestinal segment with a uniform urothelial layer.This new lining segment was durable without regrowth ofcolonic mucosa after 6 months. The new reconstitutedsegment absorbs and secretes significantly less than controlcolocystoplasty.

Conflict of interest

None.

Funding

The project described was supported by the National Cen-ter for Research Resources and the National Center for

f bladder urothelial and smooth muscle cells onto demucosalizedunctional pilot study, Journal of Pediatric Urology (2015), http://

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Advancing Translational Sciences, National Institutes ofHealth, through Grant UL1 TR000153. The content is solelythe responsibility of the authors and does not necessarilyrepresent the official views of the NIH.

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f bladder urothelial and smooth muscle cells onto demucosalizedunctional pilot study, Journal of Pediatric Urology (2015), http://