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Biomaterials 28 (2007) 1061–1070

www.elsevier.com/locate/biomaterials

Development and characterisation of a full-thickness acellularporcine bladder matrix for tissue engineering

Fiona Bollanda, Sotiris Korossisb, Stacy-Paul Wilshawc, Eileen Inghamb,c,John Fisherb, John N. Kearneyd, Jennifer Southgatea,�

aJack Birch Unit of Molecular Carcinogenesis, Department of Biology, University of York, Heslington, York YO10 5YW, UKbBiomedical Engineering Research Centre, School of Mechanical Engineering, University of Leeds, Leeds LS2 9JT, UKcInstitute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK

dTissue Services, National Blood Service, 6 Estuary Banks, Speke, Liverpool L24 8RB, UK

Received 14 July 2006; accepted 13 October 2006

Available online 7 November 2006

Abstract

The aim of this study was to produce a natural, acellular matrix from porcine bladder tissue for use as a scaffold in developing a tissue-

engineered bladder replacement. Full-thickness, intact porcine bladders were decellularised by distention and immersion in hypotonic

buffer containing 0.1% (w/v) SDS and nuclease enzymes. Histological analysis of the resultant matrices showed they were completely

acellular; that the major structural proteins had been retained and that there were some residual poorly soluble intracellular proteins. The

amount of DNA per mg dry weight of fresh porcine bladder was 2.8 (70.1) mg/mg compared to 0.1 (70.1) mg/mg in decellularised

bladder and biochemical analysis showed proportional differences in the hydroxyproline and glycosaminoglycan content of the tissue

before and after decellularisation. Uniaxial tensile testing indicated that decellularisation did not significantly compromise the ultimate

tensile strength of the tissue. There was, however, an increase in the collagen and elastin phase slopes indicating decreased extensibility.

Cytotoxicity assays using porcine smooth muscle cell cultures excluded the presence of soluble toxins in the biomaterial.

In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the urinary bladder

has been successfully developed. The matrix is biocompatible with bladder-derived cells and has potential for use in urological surgery

and tissue-engineering applications.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Bladder tissue engineering; Mechanical properties; Scaffold; Smooth muscle cells

1. Introduction

A variety of congenital and acquired conditions result incompromised bladder capacity and compliance. Currently,the major surgical solution is enterocystoplasty, wherebythe bladder is reconstructed using vascularised segments ofbowel. Although this intervention can improve continence,it is nonetheless associated with significant clinical compli-cations, as the intestine is lined by an absorptive andmucus-secreting epithelium that is incompatible with long-term exposure to urine [1]. The lack of an entirelysatisfactory clinical procedure has led researchers to pursue

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.10.005

ing author. Tel.: +44 190 432 8705; fax: +44 190 432 8704.

ess: js35@york.ac.uk (J. Southgate).

alternative strategies involving tissue engineering [2,3].Such strategies include composite enterocystoplasty,whereby the epithelium of the bowel is replaced withautologous in vitro-propagated urothelial cells [1,4,5], theincorporation of cell-seeded scaffolds [3] and the implanta-tion of synthetic, natural and composite materials into thebladder to facilitate repair and regeneration.In terms of materials for bladder reconstruction, the use

of synthetic polymers, such as polyglycolic acid, poly-ethylene and polyvinyl [6,7], has resulted in graft failureassociated with recurrent urinary tract infections, theformation of calculi, contracture of grafts and graftrejection [6]. Some clinical success has been reported fromimplanting tissue constructs prepared by seeding bladder-derived cells into composite matrices of collagen and

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Fig. 1. Photomicrograph of intact porcine bladder distended with up to

500ml of solution prior to immersion (A). Clamping ensures no loss of

liquid, so that the bladder remains distended throughout incubation in

each of the decellularisation stages. At the end of the procedure, the

sterile, decellularised bladder is dissected and the matrix presented as a

flattened sheet (B).

F. Bolland et al. / Biomaterials 28 (2007) 1061–10701062

synthetic polyglycolic acid into patients with end-stagebladder disease [3]. There has also been considerableinterest in developing natural decellularised matrices froma variety of tissue derivations, including small intestinesubmucosa (SIS) [8–13], porcine dermis (Permacols)[14,15] and the urinary bladder itself [16–20].

As natural tissue matrices are ‘‘designed for purpose’’,they offer many potential advantages over syntheticscaffold materials, and upon implantation, should beinfiltrated and remodeled by host cells into long-livedfunctional tissue [21]. As the structure of the extracellularmatrix (ECM) is highly conserved across different species[22], the potential exists to use collagenous matrices derivedfrom xenogenic sources. The preparation of naturalmatrices commonly involves a combination of physicalmethods to delaminate layers of tissue, followed bychemical and enzymatic methods to remove cell bodiesfrom the remaining ECM [23]. Such decellularisationstrategies, designed to limit the immunogenicity of thematrix, are not always entirely successful [24].

The processes of cell attachment, migration and pro-liferation are strongly influenced by the composition andstructure of the ECM, which is unique to individual tissues[25]. Therefore, a three-dimensional environment derivedfrom bladder tissue should be most effective in supportingbladder tissue regeneration following implantation.Furthermore, as the specific function of the bladder(to expand and store urine under low pressure andallow voluntary micturition) is primarily attributable tothe unique composition, structure and organisation ofthe smooth muscle wall [26], a natural material thatretained the physical properties of full-thickness nativebladder should result in improved functionality followingimplantation.

The aim of this study was to determine whether thebladder’s specific distention properties could be harnessedto develop a natural biomaterial that retained thebiological and physical properties of native bladder tissueand would, as such, be ideally suited for use as a functionalimplant in bladder reconstruction.

2. Materials and methods

2.1. Specimen procurement

Whole bladders from male, 16-week-old commercial pigs were obtained

from a local abattoir within 4 h of slaughter and transported to the

laboratory on ice in sterile transport medium [Hanks’ balanced salt

solution (HBSS, Invitrogen, Paisley, UK) containing 10mM HEPES, pH

7.6 (Invitrogen) and 10KIU/ml Aprotinin (Trasylol, Bayer, Berkshire,

UK)] [27].

2.2. Porcine bladder decellularisation

Porcine bladder decellularisation was performed under aseptic condi-

tions. Intact bladders were washed in phosphate-buffered saline (PBS)

containing 0.1% w/v ethylene diamine tetra-acetic acid (EDTA) and

Trasylol (10KIU/ml) to inhibit protease activity. Subsequent treatments,

unless otherwise stated, were all carried out with protease inhibition.

At each stage the intact bladder was distended with up to 500ml buffer

through a funnel inserted into the bladder neck, closed with NalgeneTM

Spencer wells forceps (VWR International Ltd, Poole, UK) and fully

immersed in the same solution (Fig. 1). The bladders were decellularised

by incubating the bladder for 24 h at 4 1C in hypotonic Tris buffer (10mM

Tris, pH 8.0) followed by distension and incubation with agitation on an

orbital shaker for 24 h in 0.1% (w/v) sodium dodecyl sulphate (SDS) in

hypotonic Tris buffer at ambient temperature. Bladders were washed in

PBS without protease inhibition, before being incubated for 24 h in

50U/ml deoxyribonuclease I (Sigma, Poole, UK) and 1U/ml ribonuclease

A (Sigma, Poole, UK) in 10mM Tris-HCl, pH 7.5 with gentle agitation at

37 1C. Bladders were disinfected by incubation in 0.1% (v/v) peracetic acid

in PBS for 3 h and finally, were washed in sterile PBS once for 24 h

followed by three periods of 1 h under aseptic conditions. The resultant

material was stored in PBS at 4 1C.

2.3. Histology and microscopy

Fresh and decellularised tissue samples (nX10) were fixed in 10% (v/v)

neutral buffered formalin, dehydrated and embedded in paraffin wax.

Haematoxylin and eosin staining was used to evaluate the cellular content

and general histoarchitecture of the porcine bladders. [28].

Immunolocalisation of specific proteins was performed using an

indirect immunoperoxidase method as previously described [29,30]. Five-

micrometre dewaxed sections of fresh and decellularised bladder were

incubated with monoclonal antibodies against collagen type I (COL 1),

smooth muscle actin (SMA; 1A4), laminin (LAM89) (Sigma, Poole, UK),

collagen type IV (cIV22), vimentin (V9) and desmin (D33) (Dako, High

Wycombe, UK). Each antibody was shown to cross-react with porcine

tissue prior to use. Incubation with the primary antibody was followed by

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Pressuregauge

Tubing

Air line in

Saline

Saline

Bladder

Pressurevessel

Tubing

Fig. 2. Schematic diagram of burst testing pressure rig. The rig comprised

a pressure vessel that generated test pressures, a pressure gauge for

measuring the applied hydrostatic pressures and a container filled with

saline that accommodated the bladder under testing. An inflow of air

caused the pressurisation of the saline and subsequent filling and inflation

of the bladder.

F. Bolland et al. / Biomaterials 28 (2007) 1061–1070 1063

washing and incubation with biotinylated rabbit anti-mouse Ig (F (ab0)2

fragments (Dako, High Wycombe, UK). Bound antibody was visualised

using a 3,30-diaminobenzidene substrate (DAB) reaction catalysed by

horseradish peroxidase. Omission of the primary antibody from the

labelling protocol and the use of irrelevant primary antibodies served as

negative controls.

2.4. Biochemical analysis

Three porcine bladders were decellularised for biochemical analysis and

comparison with six fresh, untreated porcine bladders. Unless otherwise

stated, test solutions for analysis were prepared from samples of fresh and

decellularised matrix that had been freeze-dried to constant weight,

hydrolysed by incubation with 6M HCl for 4 h at 120 1C and neutralised to

pH 7 with NaOH. Following analysis, a two-sample t-test assuming

unequal variance was applied to identify significant differences (p-value of

o0.05).

2.5. Glycosaminoglycan (GAG) assay

The amount of sulphated sugars (GAGs) was determined by

dimethylmethylene blue binding [31,32]. Briefly, test solutions were

incubated with the dimethylmethylene blue solution (0.03mM, Sigma,

Poole, UK) and the absorbance read at 525 nm. The amount of GAGs was

calculated by interpolation from a standard curve prepared using

chondroitin sulphate in 0.1M phosphate buffer (pH 6.8) over a range of

concentrations.

2.6. Hydroxyproline assay

Determination of hydroxyproline content was based on methods

described elsewhere [33–36]. A range of hydroxyproline standards were

prepared using trans-4-hydroxy-L-proline in hydroxyproline assay buffer

[0.17M citric acid, 0.8% (v/v) acetic acid, 0.6M sodium acetate, 0.57M

sodium hydroxide and 20% (v/v) propan-1-ol pH 6]. Oxidation of each

standard and test solution was achieved by adding N-chloro-p-toluene-

sulphonamide sodium salt solution (Chloramine T, Sigma, Poole, UK)

followed by p-dimethylamino-benzaldehyde (Ehrlich’s reagent, Sigma,

Poole, UK) and the absorbance was read at 570 nm. The amount of

hydroxyproline present in the test samples was determined against a

standard curve. To measure the amount of denatured hydroxyproline,

fresh and decellularised tissue samples that had been freeze-dried to

constant weight were digested with a-chymotrypsin prior to analysis, as

described [37].

2.7. DNA assay

Fresh and decellularised tissue samples that had been freeze-dried to

constant weight were digested in papain buffer (125mg/ml papain, 5mM

cysteine–HCl, 5mM di-sodium EDTA in PBS) at 60 1C for 24 h, as

previously described [38,39]. Test solutions were incubated with 0.1 mg/ml

Hoechst 33258 (Sigma) and using a fluorimeter, the fluorescence was read

using excitation at 365 nm and emission at 458 nm. The amount of DNA

was calculated by interpolation from a standard curve prepared using calf

thymus DNA solubilised in Tris-buffered saline (TBS, pH 7.6) over a

range of concentrations.

2.8. Biomechanical characterisation

Tissue strips measuring 20mm� 5mm were dissected from the wall of

fresh (within 24 h of slaughter) and decellularised bladders and subjected

to low strain-rate uniaxial tensile loading to failure. The anisotropy of the

bladder wall was investigated by testing specimens along the apex-to-base

and transverse directions. Prior to testing, the thickness of the tissue strips

was measured at six points along the long axis and the average thickness

was recorded. Subsequently, the specimens were mounted onto a purpose-

built titanium holder [40] and tested in a Howden tensile machine in

physiological saline at 20 1C. The specimens were preconditioned and

loaded to failure according to the testing protocol described by Korossis

et al. [40]. The load and extension data acquired during testing were

converted to engineering stress (s) and engineering strain (e) as describedelsewhere [40].

The calculated stress–strain curves obtained for the specimens of each

group were averaged over the number of specimens in each group (n ¼ 30)

using a mathematical analysis software package (Origin v6.0, Microcal

Software Inc, Northampton, USA). The stress–strain behaviour for each

specimen was analysed by means of six parameters: the elastin (El-E) and

collagen (Col-E) phase slopes, transition stress (strans) and strain (etrans),ultimate tensile strength (UTS) and failure strain (eUTS). The biomecha-

nical parameters of the specimens in each test group were averaged, and

compared by one-way analysis of variance (ANOVA).

2.9. Suture retention strength

Tissue strips measuring 10mm� 5mm were dissected from the lateral

region of fresh and decellularised bladder walls in an apex-to-base

direction and one end clamped in a titanium holder. One polypropylene

suture (Ethicon, Livingstone, UK) with 2mm bite depth was attached to

the other end of the specimen. The suture was secured to the upper part of

the holder. Subsequently, the holder with the supporting bracket was

secured to the Howden tensile machine and the suture was pulled under

uniaxial loading at a rate of 10mm/min. The suture pull-out force was

recorded.

2.10. Burst pressure

Fresh and decellularised whole bladders were subjected to burst testing

with the aid of a burst pressure rig (Fig. 2). Air was fed into the vessel at a

rate of 20ml/s, causing pressurisation of the saline in the vessel and,

subsequently, filling of the bladder. The saline pressure was increased until

the bladder burst and the maximum pressure just prior to bursting was

recorded.

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2.11. Establishment of smooth muscle cell cultures from porcine

bladder

Whole porcine bladders were obtained from a local abattoir within 4 h

of slaughter and transported to the laboratory on ice in sterile transport

medium. Pieces (1 cm2) of bladder tissue were incubated in stripping

medium (Ca2+/Mg2+-free HBSS containing 10mM HEPES, pH 7.6,

20KIU/ml Trasylol and 0.1%, w/v EDTA) at 37 1C for 4 h to detach

urothelium from the stroma [41]. The stroma was finely minced,

partially digested by incubation for 2 h in 100U/ml collagenase type IV

(Sigma, Poole, UK) and smooth muscle cells isolated as previously

described for human smooth muscle cells [15]. Cells were cultured in

Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Paisley, UK)

supplemented with 10% (v/v) fetal bovine serum (FBS) (Harlan,

Loughborough, UK) and 1% (v/v) L-glutamine (Sigma, Poole, UK) at

37 1C in a humidified atmosphere of 10% CO2 in air. Morphological

examination and immunocytochemical labelling for SMA (detailed above)

was used to confirm cell strain identity and homogeneity of the cultures, as

described [15]. Porcine smooth muscle (PSM) cell cultures were

subcultured at confluence and maintained as finite cell lines through

at least 15 passages with no change in phenotype, or were cryopreserved

for long-term storage.

2.12. Contact cytotoxicity assay

Decellularised bladder tissue was attached to the centre of a well in a 6-

well culture plate using sterile adhesive Steri-stripss (3M, Manchester,

UK). Steri-stripss ensured that the matrix was immobilised in direct

contact with the culture plate. PSM cells (passage 2–7) were seeded into

each well at a density of 1� 104 cells/ml. As negative controls, PSM cells

were seeded into wells containing Steri-strips without matrix and into wells

without Steri-strips or matrix. Cultures were incubated at 37 1C in 95%

humidity and 10% CO2 in air for 2, 4 or 12 days. Medium was then

removed from each well and the wells washed with PBS and stained/fixed

with 1% (w/v) crystal violet (Sigma, Poole, UK) in 20% (v/v) ethanol

before visualisation by light microscopy. Micrographs were captured using

a digital camera.

Fig. 3. Histological characterisation of fresh (A, B) and decellularised (C, D) p

C) and Hoeschst 33258 (B and D). Scale bar 100mm.

2.13. Growth inhibition assay

To determine the effects of matrix-conditioned medium on cell growth,

a growth inhibition assay was performed as previously described [15].

Briefly, a 5 cm2 sheet of decellularised bladder matrix was diced and added

to 50ml DMEM. After 24 h on a shaker at 37 1C the medium was

removed, filter-sterilised through a 0.2mm filter and supplemented with

10% (v/v) FBS. As a control, non-conditioned medium was prepared in a

similar way, except for omitting the diced bladder matrix. The appropriate

conditioned or non-conditioned medium was used to replace the medium

on cells in a 96-well plate in replicates of six and the plates were incubated

at 37 1C in a humidified atmosphere of 10% CO2 in air. 3-[4,5-

dimethyl(thiazol-2yl)-3,5-diphery] tetrazolium bromide (MTT) [Sigma,

Poole, UK] assays were used to compare the biomass of PSM cells grown

in control or decellularised matrix-conditioned media on days 1, 3 and 7.

2.14. Cell seeding experiments

In order to determine whether PSM cells were able to repopulate the

decellularised tissue, suspensions of 2� 105 PSM cells (at passages 2–7) in

200ml DMEM were added to the adluminal surface of decellularised tissue

samples in 6-well plates and allowed to attach for 2 h, after which time the

wells were flooded with growth medium. As a control, 200ml of cell-freeDMEM was added to tissue samples which were flooded with growth

medium, as above, after 2 h. Seeded and non-seeded samples were

collected on days 1, 3, 7, 14 and 21 in duplicate, washed in PBS and fixed

in 10% formalin for histological assessment by haematoxylin and eosin

staining.

3. Results

3.1. Characterisation of decellularised porcine bladder

The complete decellularisation of porcine bladder wasconfirmed histologically. Compared to native bladder,matrices were completely devoid of urothelium and there

orcine bladder. Sections were stained with haematoxylin and eosin (A and

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were no cells present within the underlying tissue. This wasconfirmed by Hoechst 33258 staining of sections tovisualise double-stranded DNA (Fig. 3). By contrast,histological analysis of porcine bladder tissue that hadnot been distended during the decellularisation processindicated that cells were not removed from the bladder wall(not shown).

Fig. 4. Immunoperoxidase labelling of fresh (A, C, E and G) and decellularised

collagen type I (A and B), smooth muscle actin (C and D), vimentin (E and F)

qualitative reduction in smooth muscle actin and desmin. Scale bar 200mm.

Immunolabelling with antibodies to SMA, desmin andvimentin indicated that some poorly soluble cytoskeletalcomponents of smooth muscle were not removed by thedecellularisation process (Fig. 4). An absence of immunor-eactivity with antibodies against collagen type IV andlaminin, however, confirmed removal of the basementmembrane components (Fig. 5).

(B, D, F and H) porcine bladder. Sections labelled with antibodies against

and desmin (G and H) were counterstained with haematoxylin and show a

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Fig. 5. Immunoperoxidase labelling of fresh (A and C) and decellularised (B and D) porcine bladder. Sections labelled with antibodies against collagen

type IV (A and B) and laminin (C and D) were counterstained with haematoxylin. Scale bar 200 mm.

Fig. 6. Immunofluorescence labelling of PSM cells labelled with an antibody to smooth muscle actin (A). Hoechst 33258 was used to identify all nuclei in

an equivalent field of view to demonstrate the homogeneity of the culture (B). Scale bar 50 mm.

F. Bolland et al. / Biomaterials 28 (2007) 1061–10701066

3.2. Cytotoxicity testing

MTT assays showed that PSM cells (Fig. 6) were able togrow in both control and matrix-conditioned mediumunder standard culture conditions (Fig. 7). In addition,PSM cells grew up to the decellularised matrix with noevidence of contact inhibition (Fig. 8), providing evidencethat the decellularised matrix was not cytotoxic.

PSM cells were able to attach and form a confluentmonolayer of cells across the adluminal surface of thedecellularised matrix after 3 days in culture. There was nopenetration of cells into the matrix after 7 days; however,cells had begun to infiltrate by 14 days and after 21 dayshad infiltrated the cross-section of the sample (Fig. 9).

3.3. Biochemical assays

The amount of DNA per mg dry weight of porcinebladder tissue before and after decellularisation was2.8 (70.1) and 0.1 (70.1) mg/mg, respectively (Table 1).There was a significant decrease in the DNA contentof tissue after decellularisation (t-test; po0.001).The concentrations of hydroxyproline and GAGs per mgdry weight of porcine bladder tissue before and afterdecellularisation (Table 1) were also significantly different,with the relative proportion of each being signi-ficantly higher in the decellularised tissue (t-test;po0.001), reflecting the differential removal of othercomponents.

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0

0.2

0.4

0.6

0.8

1

1.2

0

Days

Abs

orba

nce

(570

nm

)

Controlmedium

Conditionedmedium

71 3

Fig. 7. PSM cell proliferation assessed by MTT assay over 7 days. The

graph shows that under standard culture conditions in the presence of

10% (v/v) serum, growth of PSM cells was not inhibited by medium

preconditioned by incubation with decellularised matrix (n ¼ 10).

Absorbance was measured in replicates of six and the calculated standard

error of the mean (SEM) plotted as x and y error bars.

Fig. 8. Phase-contrast micrograph showing porcine SM cells growing up

to the decellularised matrix (DM) indicating that the matrix was not

cytotoxic. Scale bar 50 mm.

Fig. 9. H&E-stained section showing porcine SM cells seeded onto the

adluminal side of the decellularised porcine bladder matrix. Compared to

the lamina propria, the collagen fibers of the adluminal side were less

dense; there was greater interfibrillar space, and the surface was flatter. (A)

After 14 days in culture, there was a single layer of cells lining the surface

of the matrix and some cells were seen to have infiltrated. (B) Further

matrix penetration had occurred after 21 days. The matrix showed no

signs of degradation. Scale bar 100mm.

F. Bolland et al. / Biomaterials 28 (2007) 1061–1070 1067

3.4. Biomechanical testing

The biomechanical properties of fresh and decellularisedbladder tissues were established by uniaxial tensile loadingto failure of bladder wall strips. Significant anisotropywas found between the apex-to-base and transversedirections. Collagen phase slope and average failure strainvalues were significantly changed following decellularisa-tion undertaken with distension of the bladder, represent-ing decreased extensibility (one-way ANOVA, pp0.05).However, the UTS of the decellularised bladder wall was

not significantly different from that of the fresh bladderwall (one-way ANOVA, pX0.05).There was no significant difference in the ability of fresh

and decellularised bladder tissue to retain sutures underforce; nor in the amount of pressure required to burstintact fresh or decellularised bladders (one-way ANOVA,pX0.05).

4. Discussion

The bladder is a mechanical low-pressure, expansiblevessel, subjected to filling and voiding cycles. As such, anybiomaterial used in bladder reconstruction must havestructural properties able to withstand the forces imposedby repeated distension and contraction. In addition, thebiomaterial must be biocompatible, non-toxic and providethe correct environment for host cell repopulation.A natural, ECM-based material derived from porcinebladder that retains the same physical and structuralfeatures as native bladder tissue, but which has had allcellular components capable of producing an immunogenicresponse removed, should be an ideal candidate for bladderaugmentation. With this in mind, a decellularised porcinebladder matrix has been produced using a modification of aheart valve decellularisation protocol described by Boothet al. [29]. The innovative stage of the process exploits theidea that the bladder is an extremely compliant organ.In its retrieved contracted state, the porcine bladder istoo thick (approximately 4–5mm) to allow efficient penetra-tion of the solutions used in the decellularisation process.However, by distending the intact full thickness bladder,solutions were able to permeate throughout the wall,resulting in complete decellularisation. The decellularisationprocess included osmotic lysis of the cells, solubilisation of

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Table 1

Biochemical characterisation of fresh and decellularised porcine bladder tissue

Component Amount (mg/mg dry weight)

Fresh bladder tissue Decellularised bladder tissue

DNA 2.8 (70.1) 0.1 (70.1)*

Hydroxyproline 46.8 (72.0) 82.0 (74.3)*

Denatured hydroxyproline 2.0 (70.1) 0.7 (70.1)*

Glycosaminoglycans 20.9 (71.7) 53.2 (73.1)*

Results are presented as mean values (795% CI).

*Indicates significant difference (Student’s t-test, po0.05).

F. Bolland et al. / Biomaterials 28 (2007) 1061–10701068

cell fragments using SDS, protease inhibitors to inhibitautolysis and nucleases to digest nuclear materials and allstages were carried out under distension, a feature that isunique to this bladder-derived acellular matrix [42].

The characteristics of the decellularised matrix werecompared to the fresh tissue. The bladder wall is organisedin three major layers: urothelium, lamina propria anddetrusor smooth muscle. The urothelium, which lines thelumen of the bladder, sits on a basement membrane thatoverlies the connective tissue matrix of the lamina propriacontaining a dense layer of randomly oriented collagen fibresin which the capillary network of the bladder is embedded[43,44]. The majority of the lamina propria is constituted bya thick layer of collagen that functions to maintain the shapeof the bladder wall and to limit its overall compliance (ratioof maximum volume divided by pressure). It has been shownthat when the lamina propria is removed, the remainder ofthe bladder wall distends significantly more under physiolo-gical pressures [45]. The detrusor muscle layer provides thecontraction during voiding, and is composed of muscle fibresof 50–150mm diameter, 20–50mm apart and interconnectedwith collagen bundles [43]. Histological analysis of thedecellularised matrix showed that despite removal of theurothelium and smooth muscle components, the underlyinghistoarchitecture was retained. As the lamina propriaremained intact, there was no statistical difference in thepressure required to burst intact decellularised bladderscompared to fresh bladders. However, biomechanical testingof the decellularised matrix showed an increase in thecollagen phase slope and decreased failure strain valuescompared to fresh bladder samples, indicating that thedecellularised matrix was stiffer than fresh tissue. Thisincrease in stiffness could be due to the removal of thesmooth muscle, or due to an alteration in the ultrastructuralproperties of the collagen fibres, not resolved by histologicalexamination. For example, it could reflect a loss of the crimpform of collagen due to the decellularisation taking placewhen the tissue was distended, as such changes have not beenobserved in tissues decellularised in a relaxed state [40,46].Alternatively, it could reflect a specific characteristic ofbladder, as other authors have reported an alteration inextensibility and stiffness of acellular porcine bladder tissuefollowing processing [16]. Further functional studies will benecessary to determine whether the decreased extensibility is

significant. Importantly, there was no difference in theultimate tensile strength of the tissue following decellularisa-tion, and in terms of its potential surgical applications, therewas no difference in the ability of the decellularised matrix toretain sutures compared to fresh bladder tissue.Biomechanical testing revealed significant anisotropy

between both fresh and decellularised bladder strips inapex-to-base and transverse directions. Since the musclebundles and collagen fibres in the bladder are nothomogeneous and not always of a predictable orientation,uniaxial testing alone cannot be used to fully describe theproperties of the bladder and characterisation of thestructure–function relationship of individual bladder com-ponents will require further investigation.Within the bladder, cells, collagen (comprised of hydro-

xyproline, proline and glycine) and elastin fibres areembedded in a ground substance of GAGs. Compared tofresh bladder tissue, the proportion of hydroxyproline indecellularised tissue samples relative to total dry weightwas significantly higher, probably due to the loss of othersoluble proteins and cell components. By contrast, theproportion of denatured hydroxyproline decreased, indi-cating that the decellularisation process did not adverselyaffect the collagen content of the tissue. There was also arelative increase in the proportion of GAG in thedecellularised tissue; as GAGs bind growth factors andcytokines and promote the retention of water, the presenceof naturally occurring GAGs within the decellularisedmatrix could therefore act as substrates for cell growth.The decellularised matrices were washed extensively at

37 1C in PBS to remove any residual chemical andbiological agents. Cytotoxicity assays showed that cellsgrew well both in direct contact with the matrix and inmatrix-conditioned growth medium, indicating the wash-ing procedure was successful. Disinfection of the tissueusing peracetic acid did not affect biocompatibility, ashomologous smooth muscle cells seeded onto the adlum-inal side of the matrix had begun to repopulate the matrixafter 21 days in culture.

5. Conclusions

A novel method to produce a decellularised, biocompa-tible full-thickness bladder matrix has been successfully

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developed that exploits the bladder’s unique capacity forexpansion [42]. As the matrix retains the properties ofnative bladder tissue, it has multiple potential applicationsas a relevant scaffold for bladder engineering, as acompliant material for bladder surgery and as a researchtool for investigating cell:cell and cell:matrix interactions inbladder tissue development. The bladder is an organ thatsustains considerable structural deformation during itsnormal function; however, the role of biomechanics in thedevelopment and maintenance of the structural andbiological properties of the tissue has been little studied[26]. Understanding the material properties of the under-lying matrix contributes to this knowledge and raises thequestion as to whether biomechanical simulation maycontribute to tissue engineering goals of recellularisationand development of a functional tissue.

Acknowledgements

This work is funded by the Biotechnology and BiologicalSciences Research Council (BBSRC Grant E20352). JS isfunded by York Against Cancer.

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