Development of a Porcine Bladder Acellular Matrixwith Well-Preserved Extracellular Bioactive Factors

for Tissue Engineering

Bin Yang, M.D., Ph.D.,1,2 Yifen Zhang, M.D.,1 Liuhua Zhou, M.D.,1 Zeyu Sun, M.D.,1

Junhua Zheng, M.D.,2 Yun Chen, M.D., Ph.D.,1 and Yutian Dai, M.D., Ph.D.1

In this study, we compared four decellularization protocols and finally developed an optimized one throughwhich a porcine bladder acellular matrix (BAM) with well-preserved extracellular bioactive factors had beenprepared. In this protocol, the intact bladder was treated with trypsin/ethylenediaminetetraacetic acid to re-move the urothelium, then with hypotonic buffer and Triton X-100 in hypertonic buffer to remove the mem-branous and cytoplasmic materials, and finally with nuclease to degrade the cellular nuclear components.Bladder distention and mechanical agitation were simultaneously used to facilitate cell removal. Meanwhile,several preservative techniques, including limitation of wash time, supplement with inhibitors of proteinase,control of the pH value and temperature of the wash buffer, ethylene oxide sterilization, and lyophilization ofthe scaffold for storage, were used to protect the extracellular bioactive factors. This decellularization protocolhad completely removed the cellular materials and well preserved the extracellular collagen, sulfated glycos-aminoglycan (GAG), and bioactive factors. The preserved bioactive factors had a great potential of promotingthe proliferation and migration of both human bladder smooth muscle cell and human umbilical vein endo-thelial cell. It was also found that the amount of two representative bioactive factors, platelet-derived growthfactor BB and vascular endothelial growth factor, was positively correlated with the sulfated GAG content in theporcine BAM, implying that the amount of sulfated GAG might be a determinant for preservation of bioactivefactors in the decellularized tissues. In conclusion, the porcine BAM with well-preserved extracellular bioactivefactors might be a favorable scaffold for tissue engineering applications.

Introduction

Tissue engineering approaches used in regenerationmedicine applications firstly need to fabricate a scaffold

with bioactive factors to provide biochemical signals forregulating cell behaviors, including cell adhesion, prolifera-tion, migration, and differentiation.1 During the last decade,researchers have made great efforts to load exogenous bio-active factors into the synthetic and natural materials toimprove tissue repair.2 In spite of many promising results inthese attempts, it is still extremely difficult for biomaterialscientists to completely reproduce the extensive componentsin the native extracellular matrix (ECM) of mammals usingthe current synthetic polymers and purified biomaterials.3

Removal of cellular materials from a tissue or organ hasbeen thought an alternative approach to prepare an acellular

biological scaffold in which a complex of functional andstructural proteins, glycosaminoglycans (GAGs), glycopro-teins, and bioactive factors in the ECM can be preserved.These preserved bioactive factors are thought to have greatpotential for tissue repair and make the acellular matrix avery promising scaffold for tissue engineering.4

Bladder acellular matrix (BAM), one of the most repre-sentative decellularized tissues, has been widely used fortissue engineering of urinary bladder in various animalmodels. It had induced the ingrowth of some urothelial cells,smooth muscle cells, endothelial cells, and nerve tissues intothe scaffold from adjacent bladder parenchyma and partlyimproved bladder function after bladder augmentation.However, there were still distinguishable low-density andpoorly organized smooth muscle cells in the scaffold center.In addition, there was no rapid and adequate vascularization

1Department of Urology, Affiliated Drum Tower Hospital, Nanjing University School of Medicine, Nanjing, P.R. China.2Department of Urology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, P.R. China.

TISSUE ENGINEERING: Part CVolume 16, Number 5, 2010ª Mary Ann Liebert, Inc.DOI: 10.1089/ten.tec.2009.0311

1201

occurring in the regenerative bladder tissues, which mightlead to bladder fibrosis and eventually affect the long-termbladder function.5–12 These unsatisfactory results might beattributed to the disruption of extracellular bioactive factorsin the processes of decellularization. In these experiments,preparation of the BAM focused only on the efficiency of cellremoval, and the bladder tissues were exposed to a very highconcentration of detergents for a very long time.5–12

Recently, different decellularization techniques, includ-ing physical, chemical, and enzymatic treatments in termsof their efficiency of cell removal, effects on the ECM com-ponents, and mechanical properties of the decellularizedtissues, have been evaluated. On the basis of these investi-gations, relatively optimized decellularization protocolshave been established for the preparation of several acellu-lar matrices, including porcine skin,13 porcine anterior cru-ciate ligament,14,15 porcine heart value,16–20 rat aortic value,21

rat esophagi,22 canine ureter,23 and human amniotic mem-brane.24 To our knowledge, there was only one report ex-ploring the decellularization efficiency and mechanicalchanges of different protocols on porcine bladder tissues.25

However, current decellularized techniques are highly vari-able, and their consequent effects on the extracellular bioac-tive factors are still unknown. Further, no research has beencarried out to simultaneously consider both the efficiency ofdecellularization and preservation of extracellular bioactivefactors when the porcine BAM was prepared. The currentstudy, therefore, compared different decellularization pro-tocols on both the efficiency of cell removal and preservationof extracellular bioactive factors to develop a porcine BAMwith well-preserved extracellular bioactive factors for tissueengineering applications.

Materials and Methods

Decellularization of porcine bladder tissues

The bladder samples with in vivo capacity of about 300mLwere obtained from commercial pigs (22–25 kg) for isolationand culture of primary hepatocytes as described in thestudies of tissue engineering of liver conducted by Dr. X.H.Chu in our hospital.26 The bladder samples were immedi-ately transported in ice-cold phosphate-buffered saline (PBS;pH 7.2–7.4) to the laboratory within minutes, where the fattissues and fascia of the outside surface and the residualurine in the bladder lumen were completely removed. Thebladder tissues were then decellularized in four groups(groups A–D). In this study, all the pigs received humanecare, and the animal procedures were approved by theAnimal Care Ethics Committee of Nanjing University Schoolof Medicine and were in accordance with the institutionaland national guidelines for laboratory animals.

Group A

In group A, the bladder sample was cut into 8!10 cmpieces, and the luminal mucosa (urothelium/suburothelium)was grossly removed by surgical delamination. The resultingtissue was incubated overnight in ice-cold hypotonic Trisbuffer (10mM Tris, pH 8.0) containing 5mM ethylenedia-minetetraacetic acid (EDTA; Amersco) and 10KIU/mLaprotinin (Livzon Pharmaceutical Group Inc.). After threewashes with ice-cold PBS, the bladder was incubated for 24 h

at room temperature (RT) in hypertonic Tris buffer (50mMTris, 0.5M NaCl, 10mM EDTA, and 10KIU/mL aprotinin,pH 8.0) containing 1.0% Triton X-100 (Sigma-Aldrich).

Group B

Bladder decellularization performed in group B was sim-ilar to that in group A. After washing with Triton X-100 asabove, the bladder sample was further incubated at RT for24 h in hypertonic Tris buffer containing 1.0% sodium do-decyl sulfate (SDS) (Amersco).

Group C

In group C, a catheter was fixed at the bladder neck andthe intact bladder was decellularized using distention tech-niques modified from previous researches.27,28 Briefly, thebladder was distended through the catheter with 100mL0.25% trypsin/0.038% EDTA (Gibco), incubated in the sametrypsin/EDTA solution for 2 h at RT, and then washed thricewith ice-cold PBS. During all the following procedures, thebladder was distended with up to 300mL wash buffer andfully immersed in the same solution. The bladder was in-cubated overnight in ice-cold hypotonic Tris buffer. Afterthree washes with ice-cold PBS, the bladder was then incu-bated for 24 h at RT in hypertonic Tris buffer containing 1.0%Triton X-100.

Group D

Bladder decellularization performed in group D wassimilar to that in group C, except that the concentration ofTriton X-100 in hypertonic Tris buffer was reduced to 0.5%.

After washing with detergents in all the four groups, thebladder sample was washed thrice with ice-cold PBS, fol-lowed by incubation for 24 h at RT in 10mM Tris buffer (pH7.6) containing 50U/mL type I DNase (Sigma-Aldrich), 1U/mL Type A RNase (Sigma-Aldrich), 2mM MgCl2, 2mMCaCl2, and 150mM NaCl. During all the processes men-tioned above, except for the incubation in the ice-cold hypo-tonic Tris buffer, mechanical agitation on an orbital shaker(150 rpm; GFL) was utilized simultaneously to facilitate cellremoval.

After the decellularization procedures, the decellularizedtissue was thoroughly washed with sterile ice-cold PBS toremove the residual reagents. After three washes with steriledouble-distilled water, the decellularized tissue was frozen,lyophilized in a FreeZone 4.5 Liter Freeze Dry System(Labconco), sterilized in ethylene oxide gas, and sealed at"208C within 2 weeks.

Histological and immunohistochemical examinations

The native bladders and decellularized tissueswere fixed in10% buffered formalin, dehydrated, and embedded in paraf-fin. For histological examinations, sections (5 mm) of nativebladders and decellularized tissues were deparaffinized andstained with hematoxylin and eosin, Masson’s trichrome, and40,6-diamidino-2-phenylindole (DAPI, Molecular Probes) toevaluate the cellular nucleus and ECM components.

In immunohistochemical staining, sections were depar-affinized, blocked, and incubated at 48C overnight withmonoclonal antibodies against alpha-smooth muscle actin

1202 YANG ET AL.

(a-SMA), desmin, and vimentin (all from Sigma-Aldrich).A subsequent reaction was done using Dako REAL! EnVi-sion! Detection System, Peroxidase/DAB, and Rabbit/Mouse (Dako), and the sections were counterstained withhematoxylin. Negative controls were obtained by substitut-ing the primary antibodies with PBS.

Quantification of collagen

Freeze-dried native porcine bladders and decellularizedtissues were weighed, hydrolyzed in 6M HCl at 1108C for5 h, neutralized to pH 7 with 2M NaOH, and then subjectedto the measurements of hydroxyproline using p-dimethyl-aminobenzaldehyde (Sigma-Aldrich) and chloramine-T(Sigma-Aldrich) method.27 The absorbances at 570nm wereobtained, and the hydroxyproline levels were determinedagainst a standard curve from trans-L-hydroxyproline (Sigma-Aldrich) over a range of concentrations. The amount of totalcollagen content per mg dry weight of the BAM sample wascalculated using a hydroxyproline-to-collagen ratio of 1:7.2.

Quantification of sulfated GAG

Freeze-dried native porcine bladders and decellularizedtissues were weighed and then digested in a solution of50mg/mL proteinase K (Merck) at 568C overnight. Afterproteinase K was inactivated for 10min at 908C, the testsolution was subjected to the measurements of sulfatedGAGs by dimethylmethylene blue (Sigma-Aldrich) bindingassay with absorbances at 525 nm.29 The amounts of sulfatedGAGs were calculated against a standard curve from a rangeof chondroitin sulfate (Sigma-Aldrich) concentrations andexpressed as mg/mg dry weight of the BAM sample.

Quantification of total genomic DNA

The total genomic DNA in freeze-dried native porcinebladders and decellularized tissues was extracted usinga DNA isolation kit for tissues (Roche Applied Sciences).The samples were weighed, finely minced using scissors,and digested with proteinase K and RNase. The proteinwas precipitated and discarded. The remaining genomicDNAwas collected andquantified using theQuant-iT!PicoGreen"

dsDNA Reagent and Kit (Molecular Probes) according to themanufacturer’s protocol. The fluorescence of test solution wasmeasured on a fluorescence microplate reader (excitation,480 nm; emission, 520 nm; SpectraFluor, Tecan). The amountof DNA in the native porcine bladders and decellularizedtissues was quantified against a lDNA standard curve andexpressed as mg/mg dry weight of the test samples.

Preparation of the BAM-conditioned medium

To avoid the three-dimensional architecture of a bioma-terial on the regulation of cell behavior, the BAM-condi-tioned medium was prepared in which fetal bovine serum(FBS; Gibco) and bovine serum albumin (Gibco) were sup-plemented at a low concentration and used as carrier proteinfor many water-insoluble bioactive factors for the stabilityand the overall shelf-life of these bioactive factors. The por-cine BAM sample was suspended in 1% W/V of Dulbecco’smodified Eagle’s medium (DMEM)/F-12 (Gibco) basal me-dium (DMEM/F-12; containing 0.5% FBS and 0.5% bovineserum albumin) and endothelial basal medium-2 (EBM-2,

Lonza) basal medium (EBM-2; containing 0.5% FBS), re-spectively, and physically stirred at 48C for 7 days, afterwhich the conditioned medium was used for cell prolifera-tion and migration assays as described below. The non-conditioned medium prepared using the same lot of mediumand FBS was used as the control medium. The BAM samplesin groups B and C (five samples in each group) that had beenused for quantifications of collagen and sulfated GAG con-tents were used for preparation of the conditioned mediumand cellular biological evaluations. It was reported that thesmall intestine submucosa containing cellular residues hascytotoxic effects on urothelial cells and demonstrated obvi-ous inflammatory responses after implantation.30,31 It wasalso reported that the smooth muscle cell density graduallydecreased when the cells were cultured for a long time on theBAM with cellular materials.32 The bioactive factors mighthave been hydrolyzed and inactivated by the proteinasefrom the dead cells in the native bladder and decellularizedtissues in groups A and D.4 For these reasons, the nativebladder and decellularized tissues in groups A and D mighthave potential cytotoxic effects and had not been used forpreparation of the conditioned medium for cellular biologicalevaluation and quantification of bioactive factors.

Cell sources

In this study, both the human bladder smooth musclecell (HBSMC) and human umbilical vein endothelial cell(HUVEC) were cultured and used in cellular biological ap-proaches as described below for evaluating the extracellularbioactive factors based on the fact that bladder smoothmuscle regeneration and angiogenesis are crucial require-ments for the bladder repair and reconstruction.33 HBSMCswere cultured in DMEM/F-12 containing 10% FBS, andHUVECs were cultured in EBM-2 supplemented with theentire growth factor MV BulletKit and 10% FBS (Lonza) asour previous protocol.34 Both the cells were maintained at378C in humidified atmosphere with 5% CO2 (Direct heatCO2 incubator; Thermo Forma) and the medium was chan-ged every other day. The cell culture procedures were ap-proved by the ethics authorities of our hospital and informedconsent was obtained from each individual. Both the twoprimary cells used in the following cellular biological eval-uations were at passage 2–4.

Cell proliferation assay

HBSMCs (4!103/well) or HUVECs (5!103/well) wereplated in quintuplicate onto 0.1% gelatin (Sigma-Aldrich)-precoated 96-well plates. After the cells had attached andgrowth arrested, the medium was replaced with the controlor BAM-conditioned medium. The wells with medium onlyserved as blank control. After the cells were cultured for 48 h,10mL of Cell Counting Kit-8 (Dojindo Laboratories) wasadded into each well and the mixture was incubated foranother 3 h for HBSMC or 4 h for HUVEC. Then, the absor-bances were measured on a microplate reader (Tecan) at ameasurement/reference wavelengths at 450/620 nm.

Cell migration assay

Quantitative cell migration assay was performed usingHanging Cell Culture Inserts (Polyethylene terephthalate

DEVELOPMENT OF A PORCINE BAM WITH BIOACTIVE FACTORS 1203

filter, 8 mm pore size; Millipore) in 24-well plates (CorningInc.) as described previously.34 After serum-starvationovernight, the subconfluent HBSMCs or HUVECs were re-leased in the basal medium at 5!105/mL. After the filters ofthe inserts were coated with 0.1% gelatin for 30min at 378C,1300 mL of the control or conditioned medium per well wasadded to the bottom chambers and 200 mL of cell suspensionper well was added to the upper chambers. Two hundredmicroliters of the basal medium without cells was added tothe upper chambers for use as blank control (five wells ineach condition). After culturing for 10 h for HBSMCs or 6 hfor HUVECs, the filters were fixed and stained with 0.2%crystal violet (Sigma-Aldrich) for 20min at RT. After several

washes with water, nonmigrated cells on the upper surfaceof the filter were gently removed by a cotton-tipped swab.Then, the crystal violet in the migratory cells on the undersurface was eluted using 10% acetic acid, and the absor-bances of the blue dye mixture were read at 570 nm as ameasurement of transmigration.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) was usedfor quantification of extracellular bioactive factors. The con-trol and conditioned medium prepared in the previous sec-tions were processed using Amicon Ultra-4 Centrifugal Filter

FIG. 1. HE, Masson’s trichrome, and 40,6-diamidino-2-phenylindole (DAPI) staining of the porcine native bladder anddecellularized bladder tissues in groups A–D (scale bars in HE and DAPI staining indicate 100 and 125 mm, respectively). HE,hematoxylin and eosin. Scale bars in HE and Masson’s trichrome staining indicate 100 mm. Scale bars in DAPI stainingindicate 125 mm. Color images available online at www.liebertonline.com/ten.

1204 YANG ET AL.

Devices (3000 Nominal Molecular Weight Limit; Millipore)by which the bioactive factors with molecular weight above3 kDa can be collected and concentrated. Two representativebioactive factors, platelet-derived growth factor BB (PDGF-BB) and vascular endothelial growth factor (VEGF), in thecontrol and conditioned medium were determined by com-mercially available ELISA Kits (Uscnlife) according to themanufacture’s instructions.

Statistical analysis

All the results are presented as mean# standard devia-tion. Independent samples Student’s t-test, one-way analysisof variance followed by post hoc comparisons with theleast significant difference (LSD) test, and partial correlationanalysis with partial correlation coefficients were performedusing SPSS software (version 13.0). In all the tests, a p-valueof <0.05 was accepted as statistically significant.

Results

Evaluation of decellularization efficiency

Histological specimens of the porcine native and decel-lularized bladder tissues are shown in Figure 1. In the nativebladder, hematoxylin and eosin and Masson’s trichromestaining indicated the intact smooth muscle bundles, cellularnuclei, and collagen content in the native bladder wall. Ingroups A and D, the disturbed smooth muscle bundles withcellular nuclei can be found within a collagen ECM. Ingroups B and C, the complete elimination of cellular nucleiwas evident and porous ECM composed mainly of colla-gen fibers was preserved. DAPI staining demonstrated thedouble-stranded DNA in decellularized tissues in groups Aand D. In groups B and C, DAPI staining further confirmedthe complete elimination of cellular nuclei.

The DNA content of decellularized tissues was signifi-cantly decreased compared to that of the native porcinebladder (Table 1). There was no detectable DNA content inthe BAM samples in group B or C. The DNA content ofdecellularized tissues in group D was significantly lowerthan that in group A ( p< 0.01), implying that bladder dis-tention resulted in high decellularization efficiency eventhough the bladder tissues were exposed to a low concen-tration of detergent. This result can also be evidenced bydecellularized tissues in group C that were exposed to onlyone detergent for a short time.

Immunohistochemical examinations were further per-formed to detect the cytoskeletal materials (Fig. 2). Nativeporcine bladder was positive for a-SMA, desmin, and vi-mentin, which confirmed the cross reaction of each antibody

with porcine smooth muscle cell and fibroblast. In groups Aand D, the positive staining of a-SMA, desmin, and vimentindemonstrated that the cellular cytoskeletal components havenot been completely removed, whereas in groups B and C,the absence of staining for a-SMA, desmin, and vimentinconfirmed the complete removal of cytoplasmic materialsafter decellularization.

Cellular biological evaluation

The conditioned medium from the BAM samples ingroups B and C (n$ 5 in each group) promoted the prolif-eration of HBSMC and HUVEC (Fig. 3). The group C BAM-conditioned medium was more potent than the group BBAM-conditioned medium in stimulating proliferation ofHBSMC (% of control: 147.2%# 10.35% for group C vs.114.4%# 7.96% for group B, p< 0.01; Fig. 3A) and HUVEC(% of control: 140.6%# 8.50% for group C vs. 110.8%#11.52% for group B, p< 0.01; Fig. 3B).

These cellular biological activities were further evidencedby the enhanced migratory responses of HBSMC andHUVEC in the conditioned medium (Fig. 4). The migra-tory responses in the group C BAM-conditioned mediumwere significantly stronger than those in the group B BAM-conditioned medium for both HBSMC (% of control:133.6%# 10.24% for group C vs. 113.6%# 4.62% for group B,p< 0.01; Fig. 4A) and HUVEC (% of control: 132.8%# 7.56%for group C vs. 116.6%# 6.66% for group B, p< 0.01; Fig. 4B).

ECM components in the BAM

The results of collagen and sulfated GAG quantificationsin the native and decellularized bladder tissues are shown inTable 1. After the treatments of decellularization, a signifi-cant increase in both collagen and sulfated GAG contentswas observed, which might be due to a loss of cellular ma-terials in the decellularized tissues as identified in Figures 1and 2. The results also demonstrated that the total collagenand sulfated GAG contents in decellularized tissues in groupC were both significantly higher than those in decellularizedtissues in group B ( p< 0.05).

The results of ELISA demonstrated no detectable PDGF-BB and VEGF in the control DMEM/F-12 or EBM-2 medium.Both PDGF-BB and VEGF could be detected in the BAM-conditioned medium; thus, these bioactive factors were ex-tracted from the BAM samples. The amount of bioactivefactors extracted from the BAM samples was then expressedas pg/g dry weight of the BAM sample. Quantifications ofbioactive factors are shown in Table 2; the values indicatethat both the PDGF-BB and VEGF in the BAM samples from

Table 1. Quantification of DNA, Collagen, and Sulfated Glycosaminoglycan Contents in Nativeand Decellularized Bladder Samples

Component (mg/mg dry weight) Native (n$ 5) Group A (n$ 5) Group B (n$ 5) Group C (n$ 5) Group D (n$ 5)

DNA 2.9# 0.3 1.8# 0.2 ND ND 0.6# 0.2a

Collagen 258.1# 16.4 308.2# 11.7 676.2# 7.47 758.0# 15.7b 435.7# 11.1Sulfated GAG 24.1# 2.2 29.5# 1.7 48.3# 1.1 63.1# 4.4b 40.1# 1.7

ap< 0.01 versus group A.bp< 0.01 versus group B.ND, not detected; GAG, glycosaminoglycan.

DEVELOPMENT OF A PORCINE BAM WITH BIOACTIVE FACTORS 1205

group C were significantly higher than those in the BAMsamples from group B ( p< 0.05). The amount of bioactivefactors between the conditioned DMEM/F-12 and EBM-2medium showed no statistical difference for the BAM sam-ples from the same group ( p> 0.05).

The ECM components, including collagen, sulfated GAG,PDGF-BB, and VEGF, from group B and C BAM samples(n$ 10) were further analyzed using partial correlationanalysis to investigate the potential correlation betweencollagen (or sulfated GAG) content and bioactives factors inthe BAM samples (Table 3). The results showed that theamount of PDGF-BB and VEGF was positively correlatedwith the sulfated GAG content in the porcine BAM after

adjustment for collagen content ( p< 0.05). However, therewas no significant correlation between collagen content andbioactive factors when the effect of sulfated GAG was con-trolled ( p> 0.05).

Discussion

Efficiency of decellularization

One of the goals of a decellularization protocol is tocompletely remove cellular materials for eliminating theundesirable immune-mediated rejection after the scaffold isimplanted. In the last few decades, decellularization tech-niques have been greatly developed.4 The design of an ef-

FIG. 2. Immunohistochemical staining of the porcine native bladder and decellularized bladder tissues in groups A–D withantibodies against SMA, desmin, and vimentin (scale bars indicate 100mm). Sections were counterstained with hematoxylin.SMA, smooth muscle actin. Color images available online at www.liebertonline.com/ten.

1206 YANG ET AL.

fective decellularization protocol needs to consider the in-trinsic characteristics of a tissue or organ of interest, theunderstanding of different detergents, and the processingprograms of decellularization.35 An effective decellulariza-tion protocol should include a combination of physical,chemical, and enzymatic techniques.4

The urinary bladder is a highly compliant and distensiblevessel for reserving and voiding of the urine.36 As such,bladder distension will reduce the thickness of the bladder

wall and thus be beneficial for cell removal. Previously,bladder distention techniques were successfully used forpreparation of the porcine BAM.27,28,37 In this study, the in-tact porcine bladder was distended by wash buffer througha catheter fixed at the outlet, and our results demonstratedthat this technique had lead to a high efficiency of decel-lularization.

The urothelium of bladder forms a waterproof barrier thatprevents wash buffer from infiltrating the subepitheliallayers.36 Therefore, removal of urothelium is necessary for

FIG. 3. The BAM-conditioned medium promoted the pro-liferation of both HBSMC (A) and HUVEC (B). The group CBAM-conditioned medium was more potent than the groupB BAM-conditioned medium in stimulating proliferation ofboth HBSMC and HUVEC. *p< 0.05, #p< 0.01. BAM, bladderacellular matrix; HBSMC, human bladder smooth musclecell; HUVEC, human umbilical vein endothelial cell.

FIG. 4. The migratory activities of both HBSMC (A) andHUVEC (B) were enhanced in the BAM-conditioned me-dium. The migratory response in the group C BAM-condi-tioned medium was significantly stronger than that in thegroup B BAM-conditioned medium for both HBSMC andHUVEC. #p< 0.01.

Table 2. Detection of Bioactive Factors in the Bladder Acellular Matrix-Conditioned Medium

Bioactive factors(pg/g dry weight)

Group B Group C

DMEM/F-12 EBM-2 DMEM/F-12 EBM-2

PDGF-BB 158.4# 66.6 172.1# 71.7 594.4# 105.7a 590.7# 97.5a

VEGF 110.3# 64.1 108.8# 70.7 477.4# 88.2a 468.2# 105.0a

n$ 5 in each condition.ap< 0.01 versus group B.DMEM, Dulbecco’s modified Eagle’s medium; EBM-2, endothelial basal medium-2; PDGF-BB, platelet-derived growth factor BB; VEGF,

vascular endothelial growth factor.

DEVELOPMENT OF A PORCINE BAM WITH BIOACTIVE FACTORS 1207

subsequent removal of cells present in the smooth musclelayer. In previous researches, mechanical delamination wastried to remove the urothelium.6,10,25,38 In the processes ofmechanical delamination, the bladder wall needs to be cutinto small pieces as described in groups A and B, which willmake the bladder distension technique as described ingroups C and D unavailable. In this study, commerciallyavailable trypsin and EDTA, which have been widely usedfor isolation of cells from a tissue or organ for ex vivo culture,were used to remove the urothelium of the porcine bladder.Trypsin treatment has been used for preparation of a varietyof acellular matrices.4 Trypsin treatment was efficient to re-move the epithelial cell in the porcine skin and beneficial forthe subsequent removal of cells beneath the epithelium.13

Although the Triton X-100 is usually less effective thanSDS on decellularization,15 a combination of Triton X-100and bladder distention, hypotonic, and hypertonic bufferinduced complete removal of cellular materials in the porcinebladder wall. Physical agitation was also simultaneouslyused in this study, which is thought to be able to facilitatechemical and enzymatic exposures.4

Preservation of extracellular bioactive factors

The other goal of a decellularization protocol is to mini-mize the disruption of the biological activity of the remainingECM components. The preserved ECM components in anacellular biological scaffold would provide important bio-chemical signals for cell adhesion, proliferation, migration,and differentiation and thus be favorable for tissue repairand remodeling.4,35

Collagen, sulfated GAG, and many bioactive factors havebeen identified in the porcine BAM, including PDGF-BB,VEGF, bone morphogenetic protein 4, keratinocyte growth

factor, transforming growth factor b1 (TGFb1), insulin-likegrowth factor, basic fibroblast growth factor (bFGF), TGF-a,and epidermal growth factor (EGF). Thus, it is necessary forbiomaterial scientists to preserve these endogenous bioac-tives factors maximally in the processes of decellulariza-tion.27,39 In this study, several techniques were used toprotect the ECM components. The period of trypsin treat-ment was restricted to 2 h to avoid the decrease in ECMcomponents. The aprotinin was used to inhibit the activity ofproteases released from the disrupted cells. EDTA was alsoadded to inhibit the metalloproteases. Meanwhile, a pH of7–8 in wash buffer and relatively low temperature were usedto further limit the activity of proteases.

After decellularization, the porcine BAM was dehydratedby lyophilization, which is a well-established method com-monly used to stabilize the bioactivity of functional proteins.Lyophilization of the porcine BAM could not only make thescaffold be easily handled in the biochemical and cellularbiological evaluations, but also limit the loss of bioactivefactors during storage.35 In this study, ethylene oxide gaswas used for sterilization of the porcine BAM because eth-ylene oxide gas treatment has a slight effect on acellularbiological scaffold. The fibronectin, sulfated GAG, TGFb1,bFGF, and VEGF in the acellular matrix could be well pre-served after the sterilization of ethylene oxide gas.40 Thesebioactive factors after ethylene oxide gas treatment still re-tained biological activity and were able to support cellattachment, proliferation, and differentiation.41

Compared with the native porcine bladder, a significantincrease in both collagen and sulfated GAG contents wasobserved in the decellularized tissues, which might be due toa loss of cellular materials. In this study, the effect of twodecellularization protocols (group B vs. group C) on collagenand sulfated GAG was compared. Our results indicated thatthe amount of total collagen and sulfated GAG contentspreserved in the group C BAM was higher than that in thegroup B BAM, which was prepared by Triton X-100 and SDSin prolonged cell extraction processes. SDS can cause agreater loss of collagen and sulfated GAG contents duringdecellularization compared with Triton X-100.14,15 SDStreatment could decrease the sulfated GAG content in theacellular matrix and accordingly limit cells to re-populatethis scaffold.42

The results of cellular biological evaluations demonstratedthat the conditioned medium from the BAM samples couldpromote the proliferation and migration of HBSMC andHUVEC. These cellular responses were greater for the groupC BAM than for the group B BAM. Triton X-100 was able toleave the protein–protein interactions intact so that the bio-active factors in the group C BAM might be preserved in

Table 3. Partial Correlation Analysis of Extracellular Matrix Componentsin Porcine Bladder Acellular Matrix

Partial correlationcoefficient (r)

PDGF-BB VEGF

DMEM/F-12 EBM-2 DMEM/F-12 EBM-2

Collagen (controlling for sulfated GAG) 0.633 ( p$ 0.067) 0.654 ( p$ 0.056) 0.510 ( p$ 0.161) 0.185 ( p$ 0.634)Sulfated GAG (controlling for collagen) 0.813 ( p$ 0.008) 0.854 ( p$ 0.003) 0.847 ( p$ 0.004) 0.836 ( p$ 0.005)

p< 0.05 indicates significant correlation between the two components with the controlling variances.

Table 4. An Optimized Protocol for Preparationof the Porcine Bladder Acellular Matrix

Treatments Details

Physical Bladder distention, agitation, hypotonic, andhypertonic lysis

Chemical 1% Triton X-100Enzymatic Trypsin/EDTA, DNase, and RNaseSterilization Removal of the residual reagents and ethylene

oxide gasPreservationof ECM

Limitation of wash time, inhibition ofproteinase, control of pH value andtemperature, and lyophilization

EDTA, ethylenediaminetetraacetic acid; ECM, extracellular matrix.

1208 YANG ET AL.

functional conformations with high level of biological activ-ities. However, SDS in group B might have disrupted theprotein–protein interactions and inactivated the bioactivefactors in the resultant decellularized tissues.4

The list of the bioactive factors in the porcine BAM is veryextensive, which makes it extremely difficult to detect each ofthem. PDGF-BB and VEGF, two representative bioactivefactors, which have the ability of regulating the behavior ofsmooth muscle cells and endothelial cells, respectively, arethe two most abundant bioactive factors in the porcineBAM.39 The quantity of PDGF-BB and VEGF preserved inthe porcine BAM might be very essential for bladder smoothmuscle regeneration and angiogenesis, which are thought tobe the crucial requirements for the bladder repair and re-construction.33 For these reasons, PDGF-BB and VEGF in theporcine BAM were determined and the results further con-firmed that these two bioactive factors were better preservedin the group C BAM than in the group B BAM.

Partial correlation analysis was performed to identifywhichcomponent (collagen or sulfated GAG) was a determinant forthe preservation of bioactive factors in the decellularized tis-sues. The results indicated that only the sulfated GAG contentwas significantly correlated with the amount of bioactives inthe BAM. The sulfated GAG can bind and protect many en-dogenous bioactive factors in the native ECM.43,44 Reducingsulfated GAG content in the group B BAM might result in adecrease in bioactive factors. In addition, this study showedthat 25% decrease in sulfated GAG content resulted in about70% decrease in bioactive factors in the porcine BAM. Thisimplies that the decrease in bioactive factors was not linearrelated to the decrease in sulfated GAG content in the BAM.45

SDS treatment might not only led to a decrease in sulfatedGAG content in the process of decellularization,42 but alsoled to an alteration of conformational structure of the sulfatedGAG, which results in a low binding affinity between sulfatedGAG and bioactive factors.4,46

High level of sulfated GAG preserved in the porcine BAMmight also serve as a reservoir for delivering exogenous bio-active factors. Several researchers have tried to load exoge-nous angiogenic factors (such as bFGF and VEGF) into theBAM to improve bladder regeneration and vasculariza-tion.38,47,48 The acellular biological scaffold containing moresulfated GAGwas able to bind a greater amount of bFGF andinduce the ingrowth of a higher density of microvessels.45 Thegroup C BAM, therefore, might be more potent for loadingexogenous bioactive factors than the group B BAM.

Our study indicated no significant correlation between thecollagen content and the endogenous bioactive factors in theporcine BAM. One reason might be that there is only a verylow affinity existing between collagen fibers and bioactivefactors.49,50 This finding suggested that a reduction in colla-gen content might not lead to a significant decrease in bio-active factors. However, the reduction in collagen content inSDS treatment might result in the alteration of the ECMstructure and disruption of the mechanical integrity of thescaffold.51,52

Conclusion

An optimized protocol, a combination of physical, che-mical, enzymatic, and preservative techniques, for prepara-tion of the porcine BAM has been successfully established

(Table 4). In this protocol, the intact bladder was treated withtrypsin/EDTA to remove the urothelium, then with hypo-tonic buffer and Triton X-100 in hypertonic buffer to removethe membranous and cytoplasmic materials, and finally withnuclease to degrade the cellular nuclear components. Bladderdistention and mechanical agitation were simultaneouslyperformed to facilitate cell removal. Meanwhile, several pre-servative techniques, including limitation of wash time,supplement with inhibitors of proteinase, control of the pHvalue and temperature of the wash buffer, ethylene oxidesterilization, and lyophilization of the scaffold for storage,were used to protect the extracellular bioactive factors. Thisdecellularization protocol could completely remove the cel-lular materials and well preserve the extracellular bioactivefactors with biological activities of regulating cell behavior.These encouraging results indicated that this porcine BAMwith well-preserved extracellular bioactive factors would be afavorable scaffold for tissue engineering. Further studies ex-amining the effects of different decellularization protocols onthe collagen fiber alignment, pore architecture, and eventualbiomechanical characteristics of the porcine BAM should beconducted. Investigation of the local and systematical effect ofthe BAM on the host after implantation is also beneficial.

Disclosure Statement

No competing financial interests exist.

References

1. Atala, A. Engineering tissues, organs and cells. J Tissue EngRegen Med 1, 83, 2007.

2. Singh, M., Berkland, C., and Detamore, M.S. Strategies andapplications for incorporating physical and chemical signalgradients in tissue engineering. Tissue Eng Part B Rev 14,341, 2008.

3. Furth, M.E., Atala, A., and Van Dyke, M.E. Smart biomate-rials design for tissue engineering and regenerative medi-cine. Biomaterials 28, 5068, 2007.

4. Gilbert, T.W., Sellaro, T.L., and Badylak, S.F. Decellulariza-tion of tissues and organs. Biomaterials 27, 3675, 2006.

5. Sutherland, R.S., Baskin, L.S., Hayward, S.W., and Cunha,G.R. Regeneration of bladder urothelium, smooth muscle,blood vessels and nerves into an acellular tissue matrix.J Urol 156, 571, 1996.

6. Piechota, H.J., Dahms, S.E., Nunes, L.S., Dahiya, R., Lue,T.F., and Tanagho, E.A. In vitro functional properties of therat bladder regenerated by the bladder acellular matrix graft.J Urol 159, 1717, 1998.

7. Probst, M., Piechota, H.J., Dahiya, R., and Tanagho, E.A.Homologous bladder augmentation in dog with the bladderacellular matrix graft. BJU Int 85, 362, 2000.

8. Reddy, P.P., Barrieras, D.J., Wilson, G., Bagli, D.J., McLorie,G.A., Khoury, A.E., and Merguerian, P.A. Regeneration offunctional bladder substitutes using large segment acellularmatrix allografts in a porcine model. J Urol 164, 936, 2000.

9. Brown, A.L., Farhat, W., Merguerian, P.A., Wilson, G.J.,Khoury, A.E., and Woodhouse, K.A. 22 Week assessment ofbladder acellular matrix as a bladder augmentation materialin a porcine model. Biomaterials 23, 2179, 2002.

10. Cayan, S., Chermansky, C., Schlote, N., Sekido, N., Nunes,L., Dahiya, R., and Tanagho, E.A. The bladder acellularmatrix graft in a rat chemical cystitis model: functional andhistologic evaluation. J Urol 168, 798, 2002.

DEVELOPMENT OF A PORCINE BAM WITH BIOACTIVE FACTORS 1209

11. Obara, T., Matsuura, S., Narita, S., Satoh, S., Tsuchiya, N.,and Habuchi, T. Bladder acellular matrix grafting regener-ates urinary bladder in the spinal cord injury rat. Urology68, 892, 2006.

12. Urakami, S., Shiina, H., Enokida, H., Kawamoto, K., Kikuno,N., Fandel, T., Vejdani, K., Nunes, L., Igawa, M., Tanagho,E.A., and Dahiya, R. Functional improvement in spinal cordinjury-induced neurogenic bladder by bladder augmenta-tion using bladder acellular matrix graft in the rat. World JUrol 25, 207, 2007.

13. Chen, R.N., Ho, H.O., Tsai, Y.T., and Sheu, M.T. Processdevelopment of an acellular dermal matrix (ADM) for bio-medical applications. Biomaterials 25, 2679, 2004.

14. Harrison, R.D., and Gratzer, P.F. Effect of extraction proto-cols and epidermal growth factor on the cellular repopula-tion of decellularized anterior cruciate ligament allografts. JBiomed Mater Res A 75, 841, 2005.

15. Woods, T., and Gratzer, P.F. Effectiveness of three extractiontechniques in the development of a decellularized bone-ante-rior cruciate ligament-bone graft. Biomaterials 26, 7339, 2005.

16. Kasimir, M.T., Rieder, E., Seebacher, G., Silberhumer, G.,Wolner, E., Weigel, G., and Simon, P. Comparison of dif-ferent decellularization procedures of porcine heart valves.Int J Artif Organs 26, 421, 2003.

17. Rieder, E., Kasimir, M.T., Silberhumer, G., Seebacher, G.,Wolner, E., Simon, P., and Weigel, G. Decellularizationprotocols of porcine heart valves differ importantly in effi-ciency of cell removal and susceptibility of the matrix torecellularization with human vascular cells. J Thorac Car-diovasc Surg 127, 399, 2004.

18. Schenke-Layland, K., Vasilevski, O., Opitz, F., Konig, K.,Riemann, I., Halbhuber, K.J., Wahlers, T., and Stock, U.A.Impact of decellularization of xenogeneic tissue on extra-cellular matrix integrity for tissue engineering of heartvalves. J Struct Biol 143, 201, 2003.

19. Tudorache, I., Cebotari, S., Sturz, G., Kirsch, L., Hurschler,C., Hilfiker, A., Haverich, A., and Lichtenberg, A. Tissueengineering of heart valves: biomechanical and morpho-logical properties of decellularized heart valves. J HeartValve Dis 16, 567, 2007.

20. Grauss, R.W., Hazekamp, M.G., Oppenhuizen, F., vanMunsteren, C.J., Gittenberger-de Groot, A.C., and DeRuiter,M.C. Histological evaluation of decellularised porcine aorticvalves: matrix changes due to different decellularisationmethods. Eur J Cardiothorac Surg 27, 566, 2005.

21. Meyer, S.R., Chiu, B., Churchill, T.A., Zhu, L., Lakey, J.R.,and Ross, D.B. Comparison of aortic valve allograft decel-lularization techniques in the rat. J Biomed Mater Res A 79,254, 2006.

22. Ozeki, M., Narita, Y., Kagami, H., Ohmiya, N., Itoh, A.,Hirooka, Y., Niwa, Y., Ueda, M., and Goto, H. Evaluation ofdecellularized esophagus as a scaffold for cultured esopha-geal epithelial cells. J Biomed Mater Res A 79, 771, 2006.

23. Narita, Y., Kagami, H., Matsunuma, H., Murase, Y., Ueda,M., and Ueda, Y. Decellularized ureter for tissue-engineeredsmall-caliber vascular graft. J Artif Organs 11, 91, 2008.

24. Hopkinson, A., Shanmuganathan, V.A., Gray, T., Yeung,A.M., Lowe, J., James, D.K., and Dua, H.S. Optimization ofamniotic membrane (AM) denuding for tissue engineering.Tissue Eng Part C Methods 14, 371, 2008.

25. Rosario, D.J., Reilly, G.C., Ali Salah, E., Glover, M., Bullock,A.J., and Macneil, S. Decellularization and sterilization ofporcine urinary bladder matrix for tissue engineering in thelower urinary tract. Regen Med 3, 145, 2008.

26. Chu, X.H., Shi, X.L., Feng, Z.Q., Gu, J.Y., Xu, H.Y., Zhang,Y., Gu, Z.Z., and Ding, Y.T. In vitro evaluation of a multi-layer radial-flow bioreactor based on galactosylated chitosannanofiber scaffolds. Biomaterials 30, 4533, 2009.

27. Bolland, F., Korossis, S., Wilshaw, S.P., Ingham, E., Fisher, J.,Kearney, J.N., and Southgate, J. Development and char-acterisation of a full-thickness acellular porcine bladdermatrix for tissue engineering. Biomaterials 28, 1061, 2007.

28. Farhat, W., Chen, J., Erdeljan, P., Shemtov, O., Courtman,D., Khoury, A., and Yeger, H. Porosity of porcine bladderacellular matrix: impact of ACM thickness. J Biomed MaterRes A 67, 970, 2003.

29. Barbosa, I., Garcia, S., Barbier-Chassefiere, V., Caruelle, J.P.,Martelly, I., and Papy-Garcia, D. Improved and simple mi-cro assay for sulfated glycosaminoglycans quantification inbiological extracts and its use in skin and muscle tissuestudies. Glycobiology 13, 647, 2003.

30. Feil, G., Christ-Adler, M., Maurer, S., Corvin, S., Re-nnekampff, H.O., Krug, J., Hennenlotter, J., Kuehs, U.,Stenzl, A., and Sievert, K.D. Investigations of urothelial cellsseeded on commercially available small intestine submu-cosa. Eur Urol 50, 1330, 2006.

31. Zheng, M.H., Chen, J., Kirilak, Y., Willers, C., Xu, J., andWood, D. Porcine small intestine submucosa (SIS) is not anacellular collagenous matrix and contains porcine DNA:possible implications in human implantation. J BiomedMater Res B Appl Biomater 73, 61, 2005. DOI: 10.1089/ten.tec.2009.0099.

32. Cheng, H.L., Islam, S.S., Loai, Y., Antoon, R., Beaumont, M.,and Farhat, W.A. Quantitative MRI assessment of matrixdevelopment in cell-seeded natural urinary bladder smoothmuscle tissue-engineered constructs. Tissue Eng Part CMethods 2009. DOI: 10.1089/ten.tec.2009.0099.

33. Kanematsu, A., Yamamoto, S., and Ogawa, O. Changingconcepts of bladder regeneration. Int J Urol 14, 673, 2007.

34. Yang, B., Zhou, L., Sun, Z., Yang, R., Chen, Y., and Dai, Y.In vitro evaluation of the bioactive factors preserved inporcine small intestinal submucosa through cellular biolog-ical approaches. J Biomed Mater Res A 93, 1100, 2010.

35. Badylak, S.F., Freytes, D.O., and Gilbert, T.W. Extracellularmatrix as a biological scaffold material: structure and func-tion. Acta Biomater 5, 1, 2009.

36. Staack, A., Hayward, S.W., Baskin, L.S., and Cunha, G.R.Molecular, cellular and developmental biology of ur-othelium as a basis of bladder regeneration. Differentiation73, 121, 2005.

37. Freytes, D.O., Badylak, S.F., Webster, T.J., Geddes, L.A., andRundell, A.E. Biaxial strength of multilaminated extracellu-lar matrix scaffolds. Biomaterials 25, 2353, 2004.

38. Youssif, M., Shiina, H., Urakami, S., Gleason, C., Nunes, L.,Igawa, M., Enokida, H., Tanagho, E.A., and Dahiya, R. Effectof vascular endothelial growth factor on regeneration ofbladder acellular matrix graft: histologic and functionalevaluation. Urology 66, 201, 2005.

39. Chun, S.Y., Lim, G.J., Kwon, T.G., Kwak, E.K., Kim, B.W.,Atala, A., and Yoo, J.J. Identification and characterization ofbioactive factors in bladder submucosa matrix. Biomaterials28, 4251, 2007.

40. Hodde, J., Janis, A., Ernst, D., Zopf, D., Sherman, D., andJohnson, C. Effects of sterilization on an extracellular matrixscaffold: part I. Composition and matrix architecture. J Ma-ter Sci Mater Med 18, 537, 2007.

41. Hodde, J., Janis, A., and Hiles, M. Effects of steriliza-tion on an extracellular matrix scaffold: part II. Bioactivity

1210 YANG ET AL.

and matrix interaction. J Mater Sci Mater Med 18,545, 2007.

42. Gratzer, P.F., Harrison, R.D., and Woods, T. Matrix alter-ation and not residual sodium dodecyl sulfate cytotoxicityaffects the cellular repopulation of a decellularized matrix.Tissue Eng 12, 2975, 2006.

43. Schonherr, E., and Hausser, H.J. Extracellular matrix andcytokines: a functional unit. Dev Immunol 7, 89, 2000.

44. Zafiropoulos, A., Fthenou, E., Chatzinikolaou, G., and Tza-nakakis, G.N. Glycosaminoglycans and PDGF signaling inmesenchymal cells. Connect Tissue Res 49, 153, 2008.

45. Lai, P.H., Chang, Y., Chen, S.C., Wang, C.C., Liang, H.C.,Chang, W.C., and Sung, H.W. Acellular biological tissuescontaining inherent glycosaminoglycans for loading basicfibroblast growth factor promote angiogenesis and tissueregeneration. Tissue Eng 12, 2499, 2006.

46. Gandhi, N.S., and Mancera, R.L. The structure of glycos-aminoglycans and their interactions with proteins. ChemBiol Drug Des 72, 455, 2008.

47. Kanematsu, A., Yamamoto, S., Noguchi, T., Ozeki, M., Ta-bata, Y., and Ogawa, O. Bladder regeneration by bladderacellular matrix combined with sustained release of exoge-nous growth factor. J Urol 170, 1633, 2003.

48. Cartwright, L., Farhat, W.A., Sherman, C., Chen, J., Babyn,P., Yeger, H., and Cheng, H.L. Dynamic contrast-enhancedMRI to quantify VEGF-enhanced tissue-engineered bladdergraft neovascularization: pilot study. J Biomed Mater Res A77, 390, 2006.

49. Kanematsu, A., Yamamoto, S., Ozeki, M., Noguchi, T., Ka-natani, I., Ogawa, O., and Tabata, Y. Collagenous matrices

as release carriers of exogenous growth factors. Biomaterials25, 4513, 2004.

50. Kanematsu, A., Marui, A., Yamamoto, S., Ozeki, M., Hirano,Y., Yamamoto, M., Ogawa, O., Komeda, M., and Tabata, Y.Type I collagen can function as a reservoir of basic fibroblastgrowth factor. J Control Release 99, 281, 2004.

51. Gilbert, T.W., Wognum, S., Joyce, E.M., Freytes, D.O., Sacks,M.S., and Badylak, S.F. Collagen fiber alignment and biaxialmechanical behavior of porcine urinary bladder derivedextracellular matrix. Biomaterials 29, 4775, 2008.

52. Lumpkins, S.B., Pierre, N., and McFetridge, P.S. A me-chanical evaluation of three decellularization methods in thedesign of a xenogeneic scaffold for tissue engineering thetemporomandibular joint disc. Acta Biomater 4, 808, 2008.

Address correspondence to:Yutian Dai, M.D., Ph.D.Department of Urology

Affiliated Drum Tower HospitalNanjing University School of Medicine

No. 321 Zhongshan RoadNanjing 210008

JiangsuP.R. China

E-mail: ytdai@hotmail.com

Received: May 5, 2009Accepted: February 19, 2010

Online Publication Date: April 30, 2010

DEVELOPMENT OF A PORCINE BAM WITH BIOACTIVE FACTORS 1211