Biomaterials 26 (2005) 529–543

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*Correspondin

8605.

E-mail addres

0142-9612/$ - see

doi:10.1016/j.bio

Bladder acellular matrix as a substrate for studying in vitro bladdersmooth muscle–urothelial cell interactions

Allison L. Browna, Tamara T. Brook-Allreda, Jennifer E. Waddella, Jacinta Whiteb,Jerome A. Werkmeisterb, John A.M. Ramshawb, D"arius J. Baglic,

Kimberly A. Woodhousea,*aDepartment of Chemical Engineering and Applied Chemistry and the Institute for Biomaterials and Biomedical Engineering, University of Toronto,

200 College Street, Toronto, Ont., Canada M5S 3E5bCSIRO Division of Molecular Science, Melbourne, Vic., Australia

cDepartment of Surgery, Division of Urology, Hospital for Sick Children, Ont., Canada

Received 4 November 2003; accepted 16 February 2004

Abstract

The objective of this study was to evaluate the ability of bladder acellular matrix (BAM) to support the individual and combined

growth of primary porcine bladder smooth muscle (SMC) and urothelial (UEC) cells. An in vitro co-culture system was devised to

evaluate the effect of UEC on (i) SMC-mediated contraction of BAM discs, and (ii) SMC invasiveness into BAM. Cells were seeded

onto BAM discs under 4 different culture conditions. Constructs were incubated for 1, 7, 14 and 28 days. Samples were then

harvested for evaluation of matrix contraction.

Immunohistochemistry (IHC) was utilized to examine cellular organization within the samples and conditioned media

supernatants analyzed for net gelatinase activity. BAM contraction was significantly increased with co-culture. The same side co-

culture configuration lead to a greater reduction in surface area than opposite side co-culture. IHC revealed enhanced SMC

infiltration into BAM when co-culture was utilized. A significant increase in net gelatinase activity was also observed with the co-

culture configuration. Enhanced infiltration and contractile ability of bladder SMCs with UEC co-culture may, in part, be due to an

increase in gelatinase activity. The influence of bladder UECs on SMC behaviour in vitro indicates that BAMmay contain some key

inductive factors that serve to promote important bladder cell–cell and cell–matrix interactions.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Bladder tissue engineering; Extracellular matrix; Smooth muscle cell; Co-culture; Matrix metalloproteinase

1. Introduction

The urinary bladder is susceptible to a variety ofpathological conditions from the time of fetal develop-ment through to adulthood. Congenital abnormalitiessuch as spina bifida and disorders such as cancer,multiple sclerosis, and myelodysplasia can lead to smallcapacity, high pressure, poorly compliant or un-stable bladders. In many patients surgical interventionin the form of augmentation cystoplasty is required,the therapeutic goals of which are to provide effectiveurine storage, preservation of renal function, conti-

g author. Tel.: +1-416-978-3060; fax: +1-416-978-

s: kas@chem-eng.utoronto.ca (K.A. Woodhouse).

front matter r 2004 Elsevier Ltd. All rights reserved.

materials.2004.02.055

nence, resistance to infection and convenient, voluntaryemptying [1]. Numerous synthetic materials andnatural tissues have been investigated for the recon-struction of functionally deficient bladders. Naturalmaterials have included bladder allografts, gastrointest-inal segments, skin, muscle, pericardium, placenta,dura, small intestinal submucosa (SIS) grafts andacellular bladder matrices [2–5]. Reconfigured bowelis the most common biological tissue used for recon-struction to date. While this tissue has successfullyallowed for functional reconstruction of the bladderthere are many associated complications relatedto the incorporation of heterotypic epithelium intothe urinary tract, including metabolic abnormalities,recurrent urinary tract infections and tumor formation[6].

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In vivo tissue-engineering technologies for bladderreconstruction frequently utilize naturally derived,biodegradable materials that are placed into the hostwithout pre-seeding of cells. The presumption is that theappropriate three dimensional, extracellular matrix(ECM) environment provided by the scaffold issufficient to effectively orchestrate the regenerativecapacity of host cells [7–10]. One example of such ascaffold is small intestinal submucosa (SIS). SIS hasbeen shown to induce site specific regeneration innumerous tissues, including blood vessels, tendon,abdominal wall, ligaments and skin and more recentlythe bladder [5, 10–13].Work in our laboratory has focused on bladder

acellular matrix (BAM) as a scaffold for in vivo bladdertissue engineering. BAM is derived from full-thicknessporcine bladder; the scaffold is produced by a detergentand enzymatic extraction process which removes allcellular constituents from the bladder extracellularmatrix. The resulting material consists primarily ofcollagen and elastin and retains the basement mem-brane, which is important for urothelial cell support [4].We have previously evaluated BAM as a bladderaugment in a porcine model, with mixed results [4].The molecular mechanisms by which stromal, epithe-

lial and extracellular components interact to create afunctional bladder following augmentation with anECM-based scaffold are not currently known. Theconcept of ‘dynamic reciprocity’ between ECM proteinsand cellular elements within a specific tissue or organhas however been highlighted in several recent studies,emphasizing the importance of such tightly regulatedinteractions in maintaining homeostasis and respondingto injury in adult tissues [7, 14–18]. The matrixmetalloproteinases (MMPs) are one family of degrada-tive enzymes released by cells in the wound environ-ment. An aberrant functioning of the regulatorymechanisms controlling MMP activation are believedto contribute to pathologic states. In chronic woundhealing environments for examples, elevated levels ofMMP2 and 9, and reduced levels of proteinaseinhibitors have been reported [19]. These two MMPs,also known as gelatinases A and B, have knownproteolytic activity towards the fibrillar collagens I andIII as well as collagen IV and elastin [20]. The roles ofthese enzymes in bladder remodeling have yet to beprecisely defined however numerous studies haveestablished a role for these enzymes in vascularpathogenesis as well as demonstrating their mitogeniceffects on vascular smooth muscle cells in vitro [21–24].In order to more fully elucidate the mechanisms

involved in the progression of bladder fibrosis we havedeveloped an in vitro bladder cell culture model utilizingBAM. The objective of this study was to evaluate theability of BAM to support the individual and combinedgrowth of primary porcine bladder smooth muscle

(SMC) and urothelial (UEC) cell types. An in vitro co-culture system was devised to evaluate the effect of UECon (i) SMC mediated contraction of BAM discs, and (ii)SMC invasiveness into BAM. Experiments were de-signed to determine whether direct cellular contact(same side co-culture) versus cellular separation (oppo-site side co-culture) significantly effected SMC beha-viour on BAM. Cell seeded constructs were incubatedfor 1, 7, 14 and 28 days; at the designated time points,samples were harvested for evaluation of contractionthrough measurements of percent reduction in surfacearea. Histological and immunohistochemical (IHC)techniques were then utilized to examine cellularorganization within the samples. The conditioned mediasupernatants were analyzed at the above time points fornet activity of matrix metalloproteinases 2 and 9.

2. Materials and methods

All chemical reagents were purchased from Sigma-Aldrich Co. Canada Ltd., unless otherwise noted. Allanimals used were handled according to the guidelinesapproved by the Canadian Council for Animal Care.

2.1. Establishment of primary porcine bladder cell

cultures

2.1.1. Smooth muscle cells

Fresh bladder specimens were obtained in a sterilefield from 20–30 kg female pigs, prior to sacrifice andplaced in serum-free M199 media, containing 2%penicillin–streptomycin. The bladder tissue was cut intosmall pieces, re-immersed in collagenase solution (2mg/ml collagenase type I) and then incubated at 37�C for40min with shaking (180 rpm). The supernatant wasaspirated and complete media added (M199, containing10% FBS, and 2% penicillin–streptomycin). Thebladder pieces were minced evenly onto 150mm tissueculture treated polystyrene dishes (Becton, Dickinson,Franklin Lakes, NJ) in a few drops of complete mediaand incubated at 37�C, 5% CO2 for 12min to promotetissue adherence. Complete media was then added andthe dishes returned to the incubator. Media wascontinually replaced every 2–3 days, until 80% con-fluence was achieved at which point the cells were splitat a ratio of 1–3 using 0.5% trypsin/5.3mM EDTAsolution. Morphological examination and immunohis-tological staining for a-smooth muscle actin (a-SMA)was used to confirm cell strain identity and homogeneityof the cultures.

2.1.2. Urothelial Cells

The protocol for culturing porcine bladder urothelialcells (UEC) was developed by modifying a protocol forthe culture of human urothelial cells [25]. Fresh bladder

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specimens were obtained in a sterile field and placed inlow calcium (0.09mM) keratinocyte serum-free media(KSFM, Life Technologies Inc., Burlington, ON)containing 2% penicillin–streptomycin. The bladderwas cut in half and immersed in sterile versene (0.1%EDTA in phosphate buffered saline (PBS)) and incu-bated at 4�C overnight [50]. The next day the bladderhalves were rinsed three times in PBS. The urothelialcells were scraped from the luminal surface of thebladder and immersed in collagenase IV solution (200units/ml) and incubated at 37�C for 10min. Followingincubation, the digest solution was centrifuged for 5minat 600 rpm. The supernatant was aspirated and completemedia added (KSFM, containing 0.3% cholera toxin,0.35% bovine pituitary extract and 0.01% epidermalgrowth factor (Life Technologies). The cell suspensionwas transferred to Primaria tissue culture flasks (Becton,Dickinson) at a concentration of approximately3� 106 cells/ml and incubated at 37�C, 5% CO2. Mediawas continually replaced every 2–3 days, until 80%confluence was achieved at which time the cells weresplit at a ratio of 1 : 2 using an initial treatment ofversene, followed by 0.05% trypsin in versene. Mor-phological examination and immunohistological stain-ing with pancytokeratin (PCK, reactive to cytokeratins5, 6, 8 and 19) antibody were used to confirm urothelialcell phenotype.

2.2. Preparation of bladder acellular matrix

Porcine bladders were acellularized according to theprotocol of Brown et al. [4]. Sodium dodecyl sulphate(SDS) residual surfactant was removed from the matrixvia a final 24 h treatment with a pH 9 tris solution(Woodhouse, unpublished data). Representative sam-ples of freshly processed matrix were evaluated usinghistology and IHC to confirm the effectiveness of theextraction process. In preparation for cell seeding, BAMdiscs were cut to fit the wells of non-tissue culturetreated 24 well plates using a 16mm stainless steelbiopsy punch and soaked in a series of 70% ethanolrinses. The discs were then re-hydrated in sterile PBScontaining 1% penicillin–streptomycin for 24 h and thenequilibrated in media for 48 h prior to seeding at 37�C,5% CO2.

2.3. Experimental design

On the day of seeding, cells at passages 2–5 weretrypsinized, counted and seeded onto BAM using one ofthe following culture conditions (cell densities representnumber of cells per cell type): (A) SMCs only (ontoadluminal side, 4� 104 cells/cm2); (B) UECs only (ontoluminal side, 4� 104 cells/cm2); (C) SMCs and UECs co-cultured on the adluminal side of BAM (2� 104 cells/cm2); and (D) SMCs and UECs co-cultured on opposite

sides of BAM (SMCs onto adluminal side, 4� 104 cells/cm2). The details of cell seeding were as follows: SMCsuspensions of the appropriate concentration wereadded to the centre of each BAM disc in groups A, Cand D. The cells were allowed to attach for 3 h, afterwhich time the constructs in group D were gently liftedfrom the underlying well and flipped. UEC suspensionswere then seeded in the same fashion onto group B, C andD discs. Following UEC attachment, wells containinggroups A, C and D were topped up with complete M199while wells containing group B were topped up withcomplete KSFM. This point was designated as time 0.Experimental controls included: 2 BAM discs per time

point received no cells and were incubated in eithercomplete M199 or KSFM, serving as negative controlsfor both the contraction assay and histology. Type Icollagen gels (Vitrogen, Cohesion Technologies, PaloAlto CA) were cultured under identical conditions asthose described above for BAM and served as positivecontrols for SMC mediated matrix contraction [26].All plates were incubated at 37�C, 5% CO2, with 50%

(i.e. 500 ml) of the culture media per sample beingreplaced every two days. At 1, 7, 14 and 28 days theplates were digitally photographed using a flat bedscanner (Canon ScanGear) and imported into ScionImage Software (NIH) for measurements of percentreduction in surface area (with respect to surface area attime 0). Histological and immunohistochemical analysiswas then performed on representative samples fromeach group/time point. A sample of conditioned mediafrom each sample well was stored at �20�C for theMMP (gelatinase) activity assay.

2.4. Histology and immunohistochemistry

2.4.1. Cell monolayers

All staining was performed at room temperature. Allantibody dilutions were performed in Dako antibodydiluting buffer (Dako Diagnostics Canada Inc.). Allrinse steps were performed with 0.1% Tween 20 in tris-buffered saline (TBS, pH 7.6) for 2� 3min. Individualcultures of smooth muscle and urothelial cells weregrown to 80% confluence in triplicate wells of tissueculture treated 24 well plates. Cells were fixed in 4%paraformaldehyde for 20min, equilibrated in TBS for5min and incubated with Dako Universal Blocker for5min.

2.4.2. a-SMA staining

Fixed cells were incubated in monoclonal mouse anti-human a-SMA antibody (1 : 100; clone 1A4, Dako) for30min. After rinsing, cells were incubated in alkaline-phosphatase-conjugated rabbit anti-mouse IgG (1:30,Dako) for 30min, rinsed and then incubated for 15minwith BCIP/NBT chromogen for staining visualization.Cells were rinsed with distilled water and then

ARTICLE IN PRESSA.L. Brown et al. / Biomaterials 26 (2005) 529–543532

coverslipped using Faramount aqueous mounting med-ium (Dako).

2.4.3. Pancytokeratin staining

Fixed cells were incubated in monoclonal mouseanti-human cytokeratin (MNF116, EPOS Reagent,Dako) for 30min. After rinsing, cells were thenincubated in AEC+high sensitivity chromogen (Dako)for 8min, rinsed with distilled water and coverslipped.Stained monolayers were examined under a lightmicroscope and photographed using a Zeiss Axiovertmicroscope.

2.4.4. Characterisation of the bladder acellular matrix

Freshly processed BAM samples were fixed in 10%neutral buffered formalin for 24 h and processed forroutine histological staining with haematoxylin andeosin (H&E) to identify cell nuclei and Masson’strichrome to examine collagen organization. For IHC,deparaffinized sections were blocked for endogenousperoxidase activity with 3% aqueous hydrogen peroxidefor 15min. Sections for a-SMA and laminin stainingwere pre-treated briefly with pepsin digestion. Allsections were then treated with Protein Blocker (SignetLaboratories Inc.) for 10min. Samples were incubatedfor 1 h with one of the following antibodies: monoclonalmouse anti-human a-SMA antibody (1:200, clone 1A4,Dako), rabbit polyclonal anti-pancytokeratin (1:2000,Dako), rabbit polyclonal anti-laminin (1:100, Sigma) atroom temperature in a moist chamber. Sections werethen incubated for 30min at room temperature witheither Signet USA Level 2 biotinylated linking antibodyor with a species-specific biotinylated secondary linkingantibody as required. For staining visualization, sectionswere incubated with Ultra Streptavidin–HorseradishPeroxidase Complex (Signet USA Level 2 labelingreagent) for 30min followed by development withNovaRed (Vector Labs) for 5–10min. Sections werelightly counterstained with Mayer’s haematoxylin,dehydrated, cleared in xylene and mounted in Permount(Fischer Scientific).In order to characterize collagen composition in the

bladder wall prior to and following extraction, immuno-fluorescent analysis of native bladder and BAM wasperformed using monoclonal antibodies (mAbs) tocollagen types I, III, and VI. Native bladder and BAMsamples were fixed with O.C.T compound and frozen inliquid nitrogen prior to sectioning. Prior to collagen Istaining, slides were pretreated with 0.01% pepsin(Worthington, Lakewood, NJ) in 0.01M acetic acidfor 2.5min at 37�C and rinsed in PBS. Slides were thenincubated in mAbs to collagen types I (mouse anti-human IgG1,k, 5D8-G9; 1:10), III (mouse anti-humanIgM,k, 2G8-B1, 1:50) and VI (mouse anti-human IgG2b,1D8-F8) [27] for 40min, rinsed and then reacted withaffinity purified, FITC-conjugated, sheep anti-mouse

antibody (1:10, Silenus Laboratories, Melbourne, Aus-tralia) for 40min. Bladder samples incubated insecondary antibody only were used as controls.

2.4.5. Cell seeded constructs

For evaluation of cellular organization within the cell-seeded BAM and collagen gel samples, formalin fixed,paraffin embedded samples were sectioned at 6 mm andstained with H&E, anti-a-SMA and anti-PCK.

2.5. SEM analysis

Native bladder and BAM samples were fixed for 24 hin 2% glutaraldehyde, frozen in liquid nitrogen andfreeze fractured on cross-section using a razor blade.Fractured samples were then dehydrated in ascendingethanol solutions and critically point dried. Driedsamples were mounted on metal stubs, and sputteredwith a 6 nm thick layer of titanium. These samples wereexamined under an S-2500 scanning electron microscope(Hitachi, Japan) and images were captured using theQuartz PCI system.

2.6. Gelatinase activity

The net activity of gelatinases A and B (MMP 2 and9) were assayed on cell culture media supernatants usingthe EnzCheks Gelatinase Assay Kit (Molecular Probes,Eugene, OR). Gelatin substrate was added to the wellsof 96-well black microplates. Conditioned media wasthen added followed by the addition of 0.1M trissolution. Collagenase purified from Clostridium histoly-

ticum was used as a control enzyme. Plates containingsamples and controls were incubated in the dark atroom temperature for 18 h and then read on a SpectraMax Gemini X/S Fluorescence microplate reader(Molecular Devices) at Excitiation/Emission wave-lengths of 485/515 nm. Sample net MMP activity wasderived from interpolation of a standard curve.

2.7. Statistical analysis

Statistical analysis was performed using a commer-cially available statistical software package (OriginScientific Graphing and Analysis Software). Two-wayanalysis of variance (ANOVA) was used to examine theeffects of culture type and time on matrix contraction.For MMP activity, ANOVA was used to examine theeffects of culture type at each time point. FollowingANOVA, multiple comparisons were performed usingTukey’s post-hoc procedure. Independent Student’s t-tests were performed to identify significant differences inMMP activity between matrix types for each culturegroup. In all tests a p value of o0.05 was regarded assignificant.

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3. Results

3.1. Primary bladder cell culture

Morphological examination and IHC staining ofprimary and subcultured bladder smooth muscle andurothelial cells repeatedly confirmed cell type andhomogeneity of the cultures. In the confluent state,SMCs displayed an elongated, spindle shaped morphol-ogy and cell borders tended to be straight and welldefined (Fig. 1A). Upon subculture, low density SMCsdisplayed multiple, emanating pseudopodia until con-tact was made with neighbouring cells, whereby theclassic spindle shape was reassumed. SMCs consistentlystained positive for a-SMA (Fig. 1B). Urothelial cellsdisplayed a characteristic small, epithelioid, cobblestonemorphology (Fig. 1C) and stained positive for PCK(Fig. 1D). Based on cellular morphology, stromal cells(fibroblasts, SMCs) were not seen in these cultures,indicating the effectiveness of the initial urothelial–stromal separation as well as the inability of stromalcells to survive in a KSF medium [25].

3.2. BAM characterization

Prior to cell seeding, the acellularity of freshlyprocessed BAM was confirmed histologically. Followingprocessing of native pig tissue, matrices were completelydevoid of urothelium (Fig. 2B), as well as detrusorsmooth muscle (Fig. 2D). No remnants of cell debriswere detected throughout the cross-sections of thescaffolds and the architecture of the extracellular matrixcollagen resembled that of native bladder (Figs. 2A andC). IHC with antibodies to a-SMA and PCK furtherconfirmed the complete removal of urothelium andsmooth muscle components (Figs. 3A–D). Positivestaining for laminin in BAM sections confirmed thepreservation of this basement membrane componentalong the bladder lumen and around extracted vascu-lature (Figs. 3E and F).Surface and freeze fracture SEM of native bladder

and BAM supported histological observations. Follow-ing extraction, the characteristic folding of a relaxedurothelium (Fig. 4A) was no longer identifiable alongthe luminal BAM surface consistent with completeacellularization. Instead, a smooth, continuous, intactsuburothelial basement membrane was exposed(Fig. 4B). When examined on cross-section, nativebladder revealed transverse and longitudinal smoothmuscle bundles within the detrusor layer (Figs. 4C andE). In BAM sections, comparable areas were devoid ofsmooth muscle and the surrounding collagen appearedintact (Figs. 4D and F).Immunofluorescent staining of native bladder with

antibodies to the fibrillar collagens type I and IIIproduced a pattern that is consistent with other reports

in the literature [28]. Collagen I localized heavily to thelamina propria region of the bladder wall (not shown)and showed modest staining in the detrusor layer(Fig. 5A), while collagen III showed intense interfasi-cular (between smooth muscle bundles) localization(Fig. 5B). Collagen VI, a microfibrillar collagen, showedintense interfasicular staining as well as pericellularlocalization (Fig. 5C) in the detrusor layer. Followingextraction, immunostaining for all three collagen typeswas retained with some alteration in staining patterns.In particular, collagen III adopted a slightly morecollapsed morphology (Fig. 5E), consistent with theremoval of smooth muscle bundles from the detrusor.Collagen VI staining adopted a more diffuse pattern,with the absence of pericellular staining (Fig. 5F).Differences in collagen staining patterns between nativebladder and BAM may also be due to an alteration inthe conformational epitopes recognized by these anti-bodies as a result of acellularization. Unlike collagensIII and VI, collagen I staining in the detrusor appearedrelatively unaltered (Fig. 5D).

3.3. Cell-seeding experiments

The typical gross appearance of cell seeded collagengels and BAM discs at 7 and 28 days post-seeding areshown in Fig. 6. Measurements of collagen gel andBAM contraction versus time for the four culturegroups are shown in Fig. 7. As expected, UECs seededalone produced a negligible level of matrix contraction.For culture groups A, C and D, time had a significanteffect on contraction. In general, collagen gels con-tracted faster than BAM discs at early time points andfor both matrices, co-culture groups C and D experi-enced significantly greater matrix contraction comparedto group A (SMCs only). On collagen gels, same side co-culture (group C) lead to significantly greater matrixcontraction than opposite side co-culture (group D). OnBAM a similar trend was observed although thedifference was not statistically significant.Histological staining demonstrated cell adherence to

BAM within 24 h. SMCs were elongated and spindleshaped, and by 7 days had formed a cohesive layer, 1–2cells in thickness. Matrix contraction was visiblemicroscopically, as evidenced by areas of matrix foldingaround central SMC clusters. At later time points thesecentral areas of contraction showed a progressiveincrease in cell number and a more exaggerated spindleshaped morphology. In some regions cell layeringreached up to 8–10 cell layers in thickness. Despiteevidence of cell growth however, minimal SMC infiltra-tion into BAM was observed. UECs also formed acohesive layer of cells on BAM and by 7 days, reached2–3 layers in thickness. The cells appeared much moretightly packed than SMCs and were more polygonal inshape. In contrast to SMCs, UECs seeded alone did not

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Fig. 1. Primary porcine bladder smooth muscle and urothelial cells grown on tissue culture treated polystyrene (passage 2). (A) SMCs unstained

showing elongated, spindle shaped morphology. (B) SMCs stained for a-smooth muscle actin. (C) UECs unstained showing cobblestonemorphology. (D) UECs stained for PCK. All images taken at 100�magnification.

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display significant cell growth over the 28 days, with celllayering reaching a maximum at 7 days.Representative IHC sections of co-cultured BAM

discs 7 days post-seeding are shown in Fig. 8. When co-cultured on opposite sides (group D) the two cell typeshad an appearance similar to that of their respectivesingle cell culture configuration (i.e. group A or B).SMCs formed a cell layer 1–2 cells in thickness, withsome infiltration evident just beneath the adluminalsurface of the matrix (Fig. 8A). On the luminal side,UECs maintained a cohesive layer with no matrixpenetration. When co-cultured on the same side (groupC) distinct cell sorting was noted. SMCs grew in anelongated fashion along the adluminal surface, whileUECs grew on top of the SMCs (Fig. 8C). This cellularorganization was maintained out to 28 days. A dramaticincrease in the thickness of the urothelial cell layer wasevident at 7 days compared to culture groups B and D(Fig. 8D). Also, SMC penetration into BAM wassignificantly greater than that observed with culturegroups A and D, with numerous cells visible throughoutthe mid-section of the matrix (Fig. 8E). This pattern of

matrix penetrance by SMCs was distinctly differentfrom the pattern of growth observed when SMCs werecultured alone on BAM.The results of the gelatinolytic assay showing net

matrix metalloproteinase 2 and 9 activity are shown inFig. 9. It is pertinent to note that the enzyme–substratereaction in this assay was continuous. Therefore, inorder to control for differences in fluorescence intensitybetween assays and thus allow for accurate comparisonsbetween matrices and culture groups, values werereported as MMP activity relative to collagen gels,group A for that particular time point. ANOVArevealed significant differences in MMP activity withrespect to both matrix type and culture group. For alltime points and culture groups, MMP activity wassignificantly elevated in the presence of BAM versuscollagen gels, with the exception of culture group B at 28days. Differences between culture groups for cell seededBAM were seen at both 7 and 14 days post-seeding. Atboth time points, MMP activity of the co-culture groupswas higher than that for SMCs alone. At 7 days, thisdifference was statistically significant for co-culture

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Fig. 2. Histological analysis of acellularization. (A) Urothelium prior to extraction, and (B) after acellularization. (C) Detrusor muscle prior to

extraction and (D) after acellularization. Cellular constituents of native bladder tissue (pink) are completely absent following extraction while matrix

collagen is preserved (blue). Masson’s Trichrome. Images taken at 100�magnification.

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group C (Fig. 9B) compared to culture group A, while at14 days the difference was significant for group D (Fig.9C) compared to group A. At all 4 time points MMPactivity for BAM culture group B (UECs alone) wassignificantly higher than group A, although thisdifference may in part be due to the presence ofepidermal growth factor (EGF) in the UEC cell culturemedia, as EGF has been shown to be an inducer ofMMP 9 in bladder cells [29]. Comparisons amongstculture groups for collagen gels revealed no significantdifferences with the exception of group B at all timepoints, which again may be due to the presence of EGF.

4. Discussion

A successfully engineered genitourinary biomaterialcapable of achieving bladder regeneration must meetseveral design criteria. The material must be conductive,i.e. have physical properties and a three dimensionalarchitecture that is conducive to the functional require-ments of the bladder wall. Mechanical properties mustbe sufficient to allow for adequate urine storage capacityat low, stable intravesical pressures. The scaffold mustprovide a barrier to urine and possess no inherentabsorptive or secretory abilities. The material must alsobe inductive, i.e. contain the necessary biologicalinformation to promote desired cellular processes. Someof these functions include attraction and interaction ofappropriate cell populations, cell migration, ingrowth of

blood vessels and site-specific differentiation of in situbladder cells [30].Bladder acellular matrix addresses the first of these

design criteria. Our previous in vivo studies havehighlighted the ability of BAM augmented bladders toprovide low-pressure reservoir function and preserva-tion of normal micturition in pigs [4,9]. However, resultsof long-term implantation (22 weeks) were less favor-able. Densely arranged collagen and disorganizedsmooth muscle bundle formation within the BAMconstruct, combined with poor urothelial coverage wereobserved. Mechanical tensile testing of explanted tissuerevealed an alteration in both the extensibility andstiffness of the graft material. Combined these resultswere indicative of a fibroproliferative tissue response [4].In order to address these outcomes, a better under-standing of the cellular and molecular mechanismscontrolling wound healing events after BAM augmenta-tion is required. Our current work has focused onestablishing techniques for culturing primary porcinebladder smooth muscle and urothelial cells on BAMwith the goal of developing an effective in vitro platformfor the study of bladder cell–cell and cell–matrixinteractions.Other investigators have performed in vitro studies

using primary bladder cell culture techniques for theexamination of smooth muscle and urothelial cellphysiology. However, the majority of these studies havebeen carried out on tissue culture polystyrene or otherrigid, three-dimensional (3-D) coated surfaces [25,

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Fig. 3. Immunohistochemical analysis of acellularization. a-smooth muscle actin staining of (A) native bladder and (B) BAM and pancytokeratin

staining of (C) native bladder and (D) BAM confirm the absence of smooth muscle and urothelial cells following extraction, while laminin staining of

(E) native bladder and (F) BAM show the preservation of basement membrane along the luminal surface of the bladder as well as around blood

vessels. All images taken at 50�magnification.

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31–33]. These substrates differ significantly from theextracellular matrix surrounding in situ bladder cells.Native bladder ECM constitutes a three-dimensionalnetwork of heterogeneous macromolecules, which pro-vide both an instructive and structural milieu for tissuespecific cell functions. Thus these 2-D studies, whilevaluable, provide limited information regarding thebehaviour of bladder cells in an in vivo context. Thework of several investigators has highlighted the limi-tations of traditional tissue culture conditions forunderstanding the in vivo structure, functions, andsignaling of cell adhesions in 3-D migratory environ-ments [34].

The present study has also illustrated the impact of a3-D in vitro environment on cellular responses. We havedemonstrated the ability of BAM to support theindividual and combined growth of porcine bladderSMCs and UECs. SMC mediated contraction of BAMdiscs and collagen gels, as well as SMC invasiveness intoBAM, was influenced by co-culture with UECs. Theseeffects were time dependent and, to some extent,dependent upon the particular co-culture configurationemployed.Immunohistochemical analysis of cell seeded BAM

constructs also revealed differences in cellular organiza-tion across the culture groups. These results are

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Fig. 4. SEM analysis of acellularization. Panels A–B: Surface SEM. (A) Native bladder showing the characteristic folding of a relaxed urothelium.

(B) Following extraction, this surface characteristic is lost; only a smooth, intact suburothelial basement membrane remains. Panels C–F: Freeze

fracture SEM. (C) Detrusor layer of the native bladder showing a large, transverse smooth muscle bundle with collagen fibres surrounding it. (D)

BAM showing the preservation of collagen around an area of smooth muscle extraction. (E) Native detrusor showing longitudinal muscle bundles

lined by similarly oriented collagen. (F) BAM showing preservation of this longitudinally oriented collagen after smooth muscle extraction.

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Fig. 5. Immunofluorescent staining of native bladder and BAM with monoclonal antibodies to collagens I, III, and VI. Collagen I shows modest

staining in the detrusor layer of native bladder (A) while collagens III (B) and VI (C) show intense interfasicular localization around smooth muscle

bundles. Following extraction, immunostaining for all three collagen types is retained (D–F) although the pattern of staining is slightly different

suggesting an alteration in the conformational epitopes recognized by the antibodies as a result of acellularization. All images taken at

100�magnification.

Fig. 6. Gross appearance of cell seeded constructs at 7 and 28 days post seeding. (A) Collagen Gels, 7 days. (B) BAM, 7days. (C) Collagen Gels, 28

days. (D) BAM, 28 days. Group A: SMCs only. Group C: same side co-culture. Group D: opposite side co-culture.

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consistent with the work of Zhang et al. who reportedthe in vitro development of a well-defined urotheliumand enhanced smooth muscle cell penetration of smallintestinal submucosal (SIS) membranes when similar co-culture configurations utilizing human cells were used[35]. Interestingly, in our study, the opposite side co-culture configuration did not produce the same effect assame side co-culture, suggesting that direct SMC-UECcontact may be important in mediating cellular growthand infiltration into BAM in vitro.

The ability of stromal cells to migrate through andsubsequently remodel an extracellular matrix is largelydependent on the controlled activity of a family of zinc-dependent endopeptidases, the matrix metalloprotei-nases (MMPs). Normal physiological processes such asfetal development, inflammation, wound healing andangiogenesis depend on the controlled activity of MMPsand their inhibitors, tissue inhibitors of matrix metallo-proteinases (TIMPs), both of which can be activated byvarious stimuli including cytokines, growth factors and

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Fig. 7. Matrix contraction vs. time for cell seeded constructs. (A) Collagen Gels. (B) BAM. Error bars represent standard deviations. N=10–14 discs

per experimental group per time point. A two-way ANOVA for time and culture group was conducted for each matrix. In all comparisons time had a

significant effect on contraction. For both matrices, co-culture groups C and D experienced significantly greater matrix contraction compared to

group A (SMCs only). On collagen gels, same side co-culture (group C) led to significantly greater matrix contraction than opposite side co-culture

(group D) (Tukey’s post-hoc, po0.05). On BAM a similar trend was observed although the differences were not statistically significant.

A.L. Brown et al. / Biomaterials 26 (2005) 529–543 539

ECM interactions. In pathological states such as cancerinvasion and metastasis, arthritis, atherosclerosis, an-eurysm and fibrosis, MMP expression can becomedysregulated leading to abnormal ECM accumulationand/or turnover [21,36]. In the context of bladderoutlet obstruction, for example, the work of Backauset al. has shown that MMPs 1, 2, and 9 are significantlydown regulated in a time-dependent fashion following

exposure of bladder smooth muscle cells to increasedhydrostatic pressures [37]. Their results support theconcept that elevated pressures may cause molecularchanges in the bladder ECM consistent with decreasedcompliance and ultimately bladder dysfunction.The role of MMPs in bladder smooth muscle cell

migration in vitro has not been extensively examined,although Southgate et al. have shown that both normal

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Fig. 8. Immunohistochemical analysis of co-cultured BAM, 7 days post-seeding. Panels A, C, E: a-SMA staining (brown) of SMCs seeded onto theadluminal side of BAM (UECs are counterstained blue). Opposite side co-culture (A) exhibits minimal SMC infiltration in contrast to same side co-

culture where multiple layers of SMCs are present on the surface of the matrix (C), while additional cells have infiltrated the cross-section of the

sample (E). Panels B, D, F: PCK staining of UECs. Opposite side co-culture supports a cohesive layer of UECs 1–2 layers thick (B). In contrast, same

side co-culture (D) supports multiple cell layers adjacent to SMCs. Beneath the matrix surface there is negligible UEC infiltration (F). All images

taken at 100�magnification.

A.L. Brown et al. / Biomaterials 26 (2005) 529–543540

and neoplastic human urothelial cells produce activeMMP 2 and 9 in vitro [38]. As well, studies suggest thatMMP 2 and 9 play a key role in the destruction of thebasement membrane of superficial urothelial carcinomas[39]. MMP 2 can degrade several sub-epithelial ECMcomponents, including collagen IV, laminin and fibro-nectin. As well, this enzyme has substrate specificity forelastin, and the fibrillar collagens [20]. MMP 9 degradescollagens types III, IV and V, elastin, entactin andvitronectin [20]. Both MMP 2 and MMP 9 have beenassociated with enhanced invasion and migration ofSMC cells across and into basement membranes andcollagen matrices, respectively [22,24]. MMP 9 expressionis transiently upregulated under conditions of injury [22].In this study, we have shown that co-culture of

bladder SMCs with UECs on BAM causes a significant

increase in net gelatinase activity compared to individualSMC culture. Enhanced SMC infiltration and contrac-tile ability within BAM when co-cultured with UECsmay, in part, be due to an increase in this activity.Several studies have shown that MMPs are essential forfibroblast-mediated collagen gel contraction, althoughthe mechanism for this effect is not fully understood[40–43]. Degradation of collagen and elastin compo-nents within BAM may expose pro-migratory sequencesand/or signals within these fragments. As well, activatedMMPs may interact with integrin binding, therebyaltering the adhesive and subsequently the contractilestate of SMCs within BAM [22].The effect of co-culture on MMP activity was not

observed when the same seeding procedures wereperformed with collagen gels, despite the fact that co-

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Fig. 9. Net matrix metalloproteinase-2 and -9 activity of cell culture media supernatants taken from BAM and collagen gel discs at 1 day (A), 7 days

(B), 14 days (C) and 28 days (D) post-seeding. At each time point, values are reported as MMP activity relative to collagen gels, group A. ANOVA

revealed significant differences with respect to both matrix type and culture group (Tukey’s post-hoc, po0.05). � indicates significant differences inMMP activity with respect to matrix type for each culture group. �� indicates significant differences in MMP activity across culture group (comparedto group A) for each matrix. N=10–14 discs per experimental group per time point. Error bars represent standard deviations.

A.L. Brown et al. / Biomaterials 26 (2005) 529–543 541

culture did have a significant impact on gel contraction.As well, net gelatinase activity was significantly lowerfor collagen gel samples compared to BAM across allfour culture configurations tested. Given the fact thatfibrous connective tissue in vivo is both dynamic andcomplex with respect to its composition, it is reasonableto expect that variations in the composition of an in vitroscaffold might involve changes in both cell adhesion andsignaling. Vitrogen is largely reconstituted-type I col-lagen (95–98%) with a minimal amount (2–5%) of typeIII collagen. In contrast, BAM is a 3-D, heterogeneousmatrix consisting of collagen types I, III, and VI. Studieshave shown that fibroblast remodeling of collagen–glycosaminoglycan sponges depends on a5b1 integrin,while fibroblast remodeling of collagen matrices doesnot [44]. Instead, a2b1 is the primary integrin involvedin both fibroblast and SMC mediated contraction ofcollagen gels [45,46]. Given these differences, it ispossible that remodeling of the BAM substrate involves

an alteration in integrin usage compared to collagengels.Studies of fibroblast-mediated collagen gel contrac-

tion have also shown that the state of cellular mechan-ical loading within a matrix determines cell phenotypeand the mechanisms used to regulate contraction [47]. Ingeneral, as mechanical loading increases, an activatedcell phenotype is adopted, characterized by increased cellproliferation, matrix synthesis and actin stress fibre andfocal adhesion formation. In contrast, fibroblasts in alow load (i.e. floating collagen matrix) environment tendto become quiescent with time and changes in interginusage can lead to the eventual onset of apoptosis [44].The collagen gels utilized in this study possessed limitedmechanical integrity compared to the BAM construct,which displayed mechanical properties comparable tonative bladder [4]. Thus, it is not surprising that thecontractile and proteolytic response of bladder SMCs tothese constructs be considerably different.

ARTICLE IN PRESSA.L. Brown et al. / Biomaterials 26 (2005) 529–543542

The identification of collagen type VI as a majorcomponent of the bladder extracellular matrix has notbeen previously reported. The structure of collagen VIconsists of a short collagenous domain flanked by 2large globular domains at the C- and N-terminal ends. Itis assembled intracellularly via a unique polymerizationprocess, in which dimers and tetramers are formed thatconstitute the building blocks of microfibrils. Thesemicrofibrils are suspected to form a flexible networkinterconnecting collagen types I, III, and proteoglycans[48]. Collagen VI is involved in cell migration anddifferentiation and may play a role in bridging cells withthe ECM [49]. In vitro, collagen VI is secreted byfibroblasts and smooth muscle cells and promotesadhesion and spreading of some cell types [50].Increased expression of type VI has been reportedin wound healing and in different diseases characterizedby stromal tissue proliferation, increased ECM synth-esis, and fibrotic transformation [48,51,52]. Giventhese reported functions, it is possible that the pre-sence of collagen VI within the BAM scaffold maybe an important factor in regulating bladder cellbehaviour. Furthermore, in future studies, collagen VIsynthesis may prove to be a useful indicator ofbladder fibroproliferative responses both in vitro andin vivo.The influence of urothelial cell co-culture on bladder

smooth muscle cell behaviour on BAM indicates thatthis matrix may contain some inductive factors thatserve to promote important bladder cell–cell and cell–matrix interactions. Other studies utilizing both in vivoand in vitro tissue recombination experiments havedemonstrated that urothelial-smooth muscle cell inter-actions are crucial to bladder smooth muscle differ-entiation and organ development [18,53]. Urotheliumhas also been shown to induce smooth muscle formationand differentiation in other organs such as the gastro-intestinal tract, uterus and prostate [54]. Of particularrelevance to this study is the work of Liu et al. whichdemonstrated that bladder urothelium induces bladdersmooth muscle growth and differentiation via bothdirect cell–cell contact and through the production ofdiffusible growth factors in vitro [55]. That opposite sideco-culture of bladder SMCs and UECs produced a lesspronounced effect on matrix contraction compared tosame side co-culture with BAM suggests that anincreased diffusion length (i.e. matrix thickness) com-pared to collagen gels may have provided a diffusionalbarrier to cytokines released from UECs, particularlygiven the static nature of the model.In conclusion, future use of the BAM co-culture

model for in vitro studies may provide a closerapproximation to the in vivo environment than conven-tional substrates and thereby facilitate the identificationof some of these inductive factors as they relate toregeneration in the bladder.

Acknowledgements

The authors would like to acknowledge K.J. Aitkenfor her technical assistance and Materials and Manu-facturing Ontario (MMO), National Science and En-gineering Research Council (NSERC) and CanadianInstitutes of Health Research (CIHR) for funding thisresearch.

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