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Journal of BiomaterialsScience Polymer EditionPublication details including instructions forauthors and subscription informationhttpwwwtandfonlinecomloitbsp20

Mapping of the distributionof significant proteins andproteoglycans in smallintestinal submucosa byfluorescence microscopyRobert E Hurst amp Rebecca B BonnerPublished online 02 Apr 2012

To cite this article Robert E Hurst amp Rebecca B Bonner (2001) Mapping of thedistribution of significant proteins and proteoglycans in small intestinal submucosaby fluorescence microscopy Journal of Biomaterials Science Polymer Edition1211 1267-1279 DOI 101163156856201753395798

To link to this article httpdxdoiorg101163156856201753395798

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J Biomater Sci Polymer Edn Vol 12 No 11 pp 1267ndash1279 (2001)Oacute VSP 2001

Mapping of the distribution of signi cant proteinsand proteoglycans in small intestinal submucosaby uorescence microscopy

ROBERT E HURST curren and REBECCA B BONNERDepartment of Urology Oklahoma University Health Sciences Center PO Box 26109Oklahoma City OK 73190 USA

Received 15 May 2001 accepted 8 August 2001

AbstractmdashBecause small intestine submucosa (SIS) is a bioscaffold for tissue regeneration we de-scribe a method to analyze the material for growth peptides and for structural molecules Immuno u-orescence methods are described for relative quanti cation of abundant structural proteins Addi-tionally a quantitative technique for comparison of the content of less abundant proteins in SISwas developed using the tyramide signal ampli cation (TSA) system that is applicable to paraf n-preserved tissue blocks Frozen sections generally shredded when cut thinly enough to permit entryand washout of reagents Five micrometer sections cut from paraf n blocks were immunolabeled forcollagen heparan sulfate proteoglycans (HSPG) FGF2 TGFmacr and VEGF Images of tissue sectionswere acquired by a linear image camera and quanti ed by densitometry after thresholding the signalto minimize nonspeci c uorescence Immunohistochemistry was used to con rm the immuno uo-rescence methods HSPG was widely distributed but concentrated in vessels FGF2 was distributeddiffusely and was associated with brous structures VEGF was distributed mainly around vesselsTGFmacr was barely detectable above background Collagen brils were distinctly present and with atwo-color uorescence system the distribution of components relative to collagen can be assessedThe anatomic structure of SIS is likely to play an important role in the regeneration of tissues andfactors in remnant vessels may facilitate penetration of the matrix along these avenues

Key words Small intestine submucosa VEGF TGFmacr FGF2 heparan sulfate proteoglycan

INTRODUCTION

Small intestine submucosa (SIS) is bioscaffold material that produces scarlessregeneration and remodeling when used to repair vascular bladder and otherinjuries [1ndash8] Virtually complete regeneration of a functional bladder followingpartial cystectomy is achieved by suturing in SIS which serves as a scaffold for

currenTo whom correspondence should be addressed E-mail robert-hurstouhscedu

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1268 R E Hurst and R B Bonner

detrusor and urothelial regeneration [3 4 6 7] How it induces such nearlycomplete regeneration is not known at a mechanistic level but it is clear that thematerial not only induces ingrowth of neighboring muscle connective tissue andurothelial cells but it also supports their eventual terminal differentiation SISmust provide the proper environment of growth-stimulatory peptides and matrixmolecules necessary to initiate programs of growth of epithelial and smooth musclecells and to induce self-assembly and differentiation [9] TGF-macr and FGF-2 areamong the growth peptides identi ed [10] However little information is availableas to how these or other substances are distributed in SIS and how consistent thedisinfected material is from batch to batch Our hypothesis is that the complexarchitecture of SIS is crucial to its function and that molecular mapping of therelationships of molecules to structures would provide crucial insights as to howSIS functions as a bioscaffold for regenerative growth

The main aim of this study was to identify and quantify at least in a comparativesense key growth and structural components in the SIS biomaterial Quantitative uorescence-image analysis (QFIA) supports quantitation of biomolecules in situin relation to morphology Quantitation is achieved by treating the cell or tissueas if it were a reaction in solution and investigating the stoichiometry of probebinding [11ndash16] Antibody or other af nity reagents are titrated to equivalenceto ensure that suf cient reagent is present to saturate the sites but that theconcentration is not so high as to produce signi cant nonspeci c binding Af nityprobes themselves are carefully evaluated to ensure stoichiometric binding willoccur Under conditions of stoichiometric binding the uorescence intensity abovebackground is proportional to the amount of target molecule The instrumentationitself is carefully adjusted and calibrated so that the optical eld of the microscopeis uniform in intensity and that stray light is negligible [11] When used toanalyze individual cells target molecules such as DNA or proteins can be measuredwith a precision and accuracy that is approximately equal to that achieved withELISA [17] These same techniques can be adapted to the measurement ofmolecules in sections of tissue or other materials such as SIS in relation tomorphologic distribution

With cytology specimens the boundaries of non-contiguous cells provide a meansfor absolute quantitation in amount per cell that is not possible with tissuesHowever relative concentrations can be mapped with the same precision withtissues as can absolute concentrations be determined in cells If two target moleculesare labeled in a sample of SIS under stoichiometric conditions using different uorophores with non-overlapping emission spectra then the ratio of uorescencesat any given point is directly proportional to the ratio of concentrations at that pointDifferences in section thickness are eliminated as a source of error In this studywe compared both frozen sections and paraf n block preservation The low levelsof some of the target proteins and the tendency of SIS to shred during microtomyrequired some modi cations of the approach The method developed for relativequantitation using ampli cation of a uorescence signal in paraf n-preserved tissue

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Molecular mapping of SIS 1269

blocks was then used to compare the amounts of several growth peptides and matrixmolecules in SIS in comparison to pig mucosa

MATERIALS AND METHODS

SIS was obtained from Cook Biotech (West Lafayette IN USA) The antibodiesused in labeling are described in Table 1

Preparation and labeling of frozen sections

Approximate 25 mm2 samples of SIS were positioned on edge in freezing cassettesand surrounded by OCT compound The cassettes were then frozen in isopentanecooled with liquid nitrogen The frozen material was then sectioned on a cryostaticmicrotome at either 5 or 20 sup1m thickness Each preparation was examined byhematoxylin and eosin (HampE) staining to ensure the proper orientation of theSIS Sections were captured on slides and labeled on an automatic stainer theBioGenex Optimax The slides were incubated with the proper primary antibodywashed and then incubated with a secondary biotinylated antibody The tertiaryreagent consisted of Alexa-488 or Texas Red-labeled avidin With proper use ofisotype-speci c secondary antibodies and monoclonal primaries double-labelingwas possible

Table 1Antibodies used in study

TGFmacr1RampD MAB1835 500 microgramsvialOncogene Sciences GF33L 100 ugOncogene Sciences PF052 1 ug

VEGFRampD MAB293 500 microgramsvialBiogenex AR360-5R

Basic FGFOncogene Sciences (AB-3) GF22 100 ugmRampD AB-233-NA 1 mgvialBiogenex AR-359-5ROncogene Sciences PC16 100 ug

FibronectinRampD RDI- bronabm

Heparan sulfateSeikagaku Amer Cat 370255 02 mg

CollagenCaltag rabbit anti pig collagen I amp III PS067 1 mlVector collagen Type I NCL-COLL-Ip 1ml

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1270 R E Hurst and R B Bonner

Preparation and labeling of parafn sections

SIS was xed for 24 h with 10 buffered formalin The xed pieces about25 mm2 in size were processed and embedded in paraf n on edge and cut into5 sup1m thickness sections that were deparaf nized hydrated and labeled with theBioGenex Optimax automatic stainer A more extensive deparaf nization than isusual was employed because we have found more extensive dewaxing decreasesnonspeci c background uorescence in tissue sections Sections were dewaxed for3 min each in three changes of xylene Each week the rst solution is discardedan a fresh third solution is prepared The sections are then washed sequentiallyin two solutions of absolute ethanol then 95 75 and 50 for 5 min followed bytwo washes against deionized water For immunohistochemical labeling the ZymedST5050 AEC detection system was used A counterstain of hematoxylin generallywas employed to visualize nuclei and tissue architecture For uorescence labelingthe tyramide signal ampli cation system (TSA NEN Inc) was used to enhance thesignal to noise ratio with uorescence Nuclei were labeled with 10 sup1M Hoechst33258 in 25 ethanol-MOPSO buffer According to literature published by NENthis method generally achieves a roughly 100-fold enhancement of the speci c uorescence signal with no increase in background

Standardization of uorescence

Originally the plan to standardize uorescence was to use collagen as a standardwith any additional components being measured with a second color Collagenis present in all SIS and a pixel-by-pixel standardization of any component tocollagen could provide a structural map However as we show below thethickness of sections necessary to prevent shredding of the SIS precluded accuratequanti cation We therefore used an alternate approach in which pig intestinewas used as an arbitrary standard against which various SIS preparations couldbe compared Images were captured using a Nikon Microphot FXA microscopeequipped with a highly linear fast Hammamatsu Color Chilled 3CCD camerathat permitted image capture before signi cant fading of uorescence occurredQuanti cation was carried out using a separate system originally developed forcytology preparations [11] Ten elds were measured for each preparation afterthresholding to remove as much of the nonspeci c signal as possible

Antigen retrieval

A number of antigen retrieval approaches were investigated to determine if theyenhanced the signal Signal enhancement might result if the target protein is boundin a tight complex that masks the epitope recognized by the antibody or is maskedby formaldehyde cross-linking Antigen retrieval is the general process by whichselective denaturation reveals masked protein antigens to the antibody TargetingUnmasking Fluid (Signet) Citra Citra Plus and AR10 (BioGenex) are products

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Molecular mapping of SIS 1271

for denaturing supramolecular complexes in tissue sections and these were used todetermine if signal could be enhanced

RESULTS

5 vs 20 sup1m sections and sectioned formaldehyde-preserved tissue blocksvs frozen sections

The general standard for histopathologic examination is the 3 or 5 sup1m section ofparaf n-embedded formaldehyde-preserved material Unless the target is highlyexpressed the paraf n section usually is unsuitable for uorescence applicationsbecause of background uorescence Consequently uorescence assays on histo-logic material typically are performed on frozen sections The material to be exam-ined is frozen at dry ice or liquid nitrogen temperatures into a block of supportivematerial which then is sectioned with a microtome For various reasons frozensections do not retain morphology nearly so well as paraf n sections displayingmore distortions from the cutting process and often displaying something knownas lsquofreeze artifactrsquo Thus the examination of tissue material very much representsachieving a balance among competing limiting factors

These competitive forces are particularly relevant in examining SIS which istough and tends to tear or shred when sectioned into 5 sup1m frozen sections Thetearing tends to destroy anatomic relationships Sections cut at 20 sup1m retainmorphology but washing out unbound reagents becomes problematic Thus carefulattention must be paid to details of speci city To assist with determining speci citya sample of pig intestine was used to evaluate antibody speci city and the adequacyof the assay because the presence of distinct epithelium submucosa and muscularisallows evaluation of speci city Figure 1A illustrates dual labeling for collagen and bronectin in a 5-sup1m section of SIS Areas that are green express heparan sulfateAreas that are red express collagen and areas that are brown express both Thisis one of the best preserved 5 sup1m sections A more usual section is shown inFig 1B which shows heparan sulfate and collagen in SIS labeled with different uorophores Although the dual labels are clearly evident the structure of the SIShas essentially been lost

Figure 1C illustrates the speci city of labeling as assessed with a sample of intactpig intestine triply labeled for DNA (blue) collagen (red) and bronectin (green)The DNA label unambiguously identi es the villous epithelium The bronectinis seen mainly in the extracellular matrix of the epithelium while the collagen isfound mainly in the submucosa This distribution illustrates the speci city of theantibodies and shows that nonspeci c binding is minimal However with the usualshredding seen in Fig 1B quantitative analysis of SIS proved dif cult

With 20-sup1m frozen sections the morphology was preserved but the resultsbecame inconsistent The limiting factor seemed to be washing out of unboundreagents Systematic investigations showed that increasing the salt concentration in

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1272 R E Hurst and R B Bonner

Figure 1 Photographs of labeled SIS and pig intestine All photomicrographs are at the samemagni cation indicated by the bar in N (A) Double uorescent label of bronectin (green) andcollagen (red) in a 5-sup1m frozen SIS section demonstrating occasional preservation of morphology(B) A more usual result showing shredding and loss of morphology in a 5-sup1m frozen SIS sectionlabeled for heparan sulfate proteoglycan (green) and collagen (red) (C) Triple label of pig intestinedemonstrating speci city of labeling for bronectin (green) collagen (red) and DNA (blue) Thegreen area on the right is the villous epithelium The brown area is the submucosa from which SISis obtained (D) Labeling of collagen in a 20 sup1m section of SIS by indirect assay (E)ndash(H) Se-quential sections of pig intestine (E F) or SIS (G H) labeled for collagen by TSA ampli cation(E G) and heparan sulfate using a conventional sandwich method (F H) with 5 sup1m sections ofparaf n-preserved blocks Note the lack of background in areas where collagen or heparan sulfateis absent even with the heparan sulfate assay which does not employ the TSA ampli cation Theboundary between the submucosa and epithelium is at the bottom of the picture (E G) (I) and (J)Immunohistochemicallabelingof pig intestine(I) and SIS (J) for heparan sulfate proteoglycanin 5 sup1mparaf n block sections (K) and (L) FGF2 labelingby uorescence(K) and immunohistochemistry(L)demonstrating diffuse brous labeling Note FGF2 seems to be concentrated on the lumenal surface(top L) (M) and (N) VEGF labeling in pig intestine (M) and SIS (N) demonstrating localization tovessels and loss from SIS

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Molecular mapping of SIS 1273

Figure 2 Titration of rabbit anticollagen (f) (positive) and (F)(negative control) in 20 sup1m frozensections of SIS using indirect immuno uorescence (sandwich) assay in comparison to TSA methodwith 5 sup1m sections The negative control substituted an unrelated serum at the same dilution as theprimary antibody The x-axis is arbitrary because the concentration of the polyclonal antibody wasunknown The difference between the positive and negative is proportional to the content of collagenin the sample The attening of the curve indicates saturation of binding sites and proportionality(s) shows the titration of the antibody using the TSA method on SIS and ( u ) on pig intestine Thedifferencebetween the two curves represents the difference in collagen immunoreactivitybetween thetwo specimens

wash buffers as well as the number of washes reduced the background uorescencebut never eliminated it In spite of these improvements consistent low backgroundcould not be obtained with the 20-sup1m sections Often speci c binding could beseen as bright areas against a low-intensity background These problems were ofleast signi cance for collagen which is highly abundant but were more signi cantfor markers that were of low abundance

Figure 2 illustrates the titration of the anti-collagen antibody with a conventionallsquosandwichrsquo assay on a 20-sup1m section of porcine intestine provided by Cook Biotechas being representative of the material from which SIS is made In the absenceof primary antibody there clearly is a background uorescence as shown bythe intercept on the y-axis The amount is the same with an unrelated primaryindicating that the background is due to failure to wash out all the uorophoreIncreasing the concentration of antibody yields the expected saturation of sites Inthe at portion of the curve the difference between background and the antibody-treated sample is directly proportional to the amount of target in this case collagen

Tyramide signal ampli cation (TSA) method

This technique offers a means to amplify the speci c signal arising from binding ofan individual antibody to its antigen in 5-sup1m paraf n sections thereby enhancingthe signal to noise ratio suf ciently to swamp out the nonspeci c uorescenceseen in paraf n sections This nonspeci c uorescence is not due to the antibody

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1274 R E Hurst and R B Bonner

because it appears in the absence of any reagents Instead of a uorochrome-labeledprimary or secondary the detection system consists of horseradish peroxidase(HRP) conjugated to the secondary antibody which then catalyzes deposition ofinsoluble biotin or dye-labeled tyramide in the immediate vicinity of the HRPAs each HRP moiety can generate numerous tyramide moieties a marked signalenhancement is observed The titration of antibody in the TSA system also isshown in Fig 2 The nonspeci c uorescence background is virtually undetectablein the controls for these assays of collagen which is a high-expression proteinand represents the ideal case for both conventional immuno uorescence assays andthe TSA ampli cation method The uorescence increases with primary antibodyconcentration from essentially zero indicating the ampli cation of the speci csignal over the nonspeci c signal As with the common lsquosandwichrsquo method thecurve breaks over indicating saturation of sites The difference in uorescenceintensity between the SIS and pig samples is proportional to the difference incollagen I content of the two samples

Heparan sulfate proteoglycans are of interest because they bind growth peptides invivo [18] The HS-PG is clearly detectable in the SIS material without ampli cation(Fig 1B F and H) Figure 3 shows the titration of heparan sulfate with theTSA method and demonstrates the presence of HS-PG in both the submucosalregion of the pig intestine and the SIS with the intestinal sample having a higherconcentration of the proteoglycan Figure 1EndashH shows the appearance of thecollagen enhanced by the TSA method and HS-PG labeled with a conventionallsquosandwichrsquo assay Collagen is labeled with red while heparan sulfate proteoglycanis labeled in green Figures 1E and F demonstrate the intact pig intestine whileFig 1G and H demonstrate SIS Note that the sections seem to be cut along differentplanes The glands and vessels are positive for heparan sulfate with very little

Figure 3 Titration of anti-heparan sulfate antibody using TSA method for SIS (s) and pigintestine (u )

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Molecular mapping of SIS 1275

background particularly in the SIS (Fig 1G and H) These high abundance proteinscould be quanti ed either from conventional immun uorescence even on paraf nsections or with the TSA method

With the TSA method negative controls are undetectable for measurement asare the lowest concentrations of this titration curve (Fig 3) Binding appears tobe very speci c and intense Both the unprocessed porcine intestine and the SISmaterial demonstrate speci c binding of anti-heparan sulfate antibody Vessels arethe primary positive component in the submucosa The values indicate that there issome loss of antigenicity (about 35) in SIS material compared to the intestinesample but clearly heparan sulfate is present within the body of the SIS itself(Fig 1H) The uorescence data are con rmed by immunohistochemical labelingwhich is demonstrated in Fig 1I and J Clearly the heparan sulfate proteoglycan ismore prevalent in the section derived from the pig than from the SIS This con rmsthe loss of some of the heparan sulfate proteoglycans during the processing

Growth peptides and low abundance components of SIS

Clearly the TSA procedure can identify and quantify components in tissues andSIS We next sought to apply it to components likely to be present in lowerconcentrations such as growth peptides which might not be evident against thebackground uorescence The distribution of FGF2 TGFmacr and VEGF in SIS wereinvestigated in relation to the amounts seen in pig intestine

Figure 4 illustrates a titration of the FGF2 antibody in the TSA system Thetwo points shown with the circle and square represent the controls which representan unrelated antibody of the same isotype as the primary used at the highestconcentration the speci c primary is used The lowest background values of thirdygray level values is still well below the speci c signal The values on the ordinate arearbitrary and are adjusted to keep the signal within the linear range of the measuring

Figure 4 Titration of anti-FGF2 antibody using TSA method for SIS (s) and pig intestine (u )(fF) represent an unrelated isotype-controlledmonoclonal antibody used as a negative control at thehighest concentrationused in the titration of the speci c antibody

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1276 R E Hurst and R B Bonner

Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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1268 R E Hurst and R B Bonner

detrusor and urothelial regeneration [3 4 6 7] How it induces such nearlycomplete regeneration is not known at a mechanistic level but it is clear that thematerial not only induces ingrowth of neighboring muscle connective tissue andurothelial cells but it also supports their eventual terminal differentiation SISmust provide the proper environment of growth-stimulatory peptides and matrixmolecules necessary to initiate programs of growth of epithelial and smooth musclecells and to induce self-assembly and differentiation [9] TGF-macr and FGF-2 areamong the growth peptides identi ed [10] However little information is availableas to how these or other substances are distributed in SIS and how consistent thedisinfected material is from batch to batch Our hypothesis is that the complexarchitecture of SIS is crucial to its function and that molecular mapping of therelationships of molecules to structures would provide crucial insights as to howSIS functions as a bioscaffold for regenerative growth

The main aim of this study was to identify and quantify at least in a comparativesense key growth and structural components in the SIS biomaterial Quantitative uorescence-image analysis (QFIA) supports quantitation of biomolecules in situin relation to morphology Quantitation is achieved by treating the cell or tissueas if it were a reaction in solution and investigating the stoichiometry of probebinding [11ndash16] Antibody or other af nity reagents are titrated to equivalenceto ensure that suf cient reagent is present to saturate the sites but that theconcentration is not so high as to produce signi cant nonspeci c binding Af nityprobes themselves are carefully evaluated to ensure stoichiometric binding willoccur Under conditions of stoichiometric binding the uorescence intensity abovebackground is proportional to the amount of target molecule The instrumentationitself is carefully adjusted and calibrated so that the optical eld of the microscopeis uniform in intensity and that stray light is negligible [11] When used toanalyze individual cells target molecules such as DNA or proteins can be measuredwith a precision and accuracy that is approximately equal to that achieved withELISA [17] These same techniques can be adapted to the measurement ofmolecules in sections of tissue or other materials such as SIS in relation tomorphologic distribution

With cytology specimens the boundaries of non-contiguous cells provide a meansfor absolute quantitation in amount per cell that is not possible with tissuesHowever relative concentrations can be mapped with the same precision withtissues as can absolute concentrations be determined in cells If two target moleculesare labeled in a sample of SIS under stoichiometric conditions using different uorophores with non-overlapping emission spectra then the ratio of uorescencesat any given point is directly proportional to the ratio of concentrations at that pointDifferences in section thickness are eliminated as a source of error In this studywe compared both frozen sections and paraf n block preservation The low levelsof some of the target proteins and the tendency of SIS to shred during microtomyrequired some modi cations of the approach The method developed for relativequantitation using ampli cation of a uorescence signal in paraf n-preserved tissue

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Molecular mapping of SIS 1269

blocks was then used to compare the amounts of several growth peptides and matrixmolecules in SIS in comparison to pig mucosa

MATERIALS AND METHODS

SIS was obtained from Cook Biotech (West Lafayette IN USA) The antibodiesused in labeling are described in Table 1

Preparation and labeling of frozen sections

Approximate 25 mm2 samples of SIS were positioned on edge in freezing cassettesand surrounded by OCT compound The cassettes were then frozen in isopentanecooled with liquid nitrogen The frozen material was then sectioned on a cryostaticmicrotome at either 5 or 20 sup1m thickness Each preparation was examined byhematoxylin and eosin (HampE) staining to ensure the proper orientation of theSIS Sections were captured on slides and labeled on an automatic stainer theBioGenex Optimax The slides were incubated with the proper primary antibodywashed and then incubated with a secondary biotinylated antibody The tertiaryreagent consisted of Alexa-488 or Texas Red-labeled avidin With proper use ofisotype-speci c secondary antibodies and monoclonal primaries double-labelingwas possible

Table 1Antibodies used in study

TGFmacr1RampD MAB1835 500 microgramsvialOncogene Sciences GF33L 100 ugOncogene Sciences PF052 1 ug

VEGFRampD MAB293 500 microgramsvialBiogenex AR360-5R

Basic FGFOncogene Sciences (AB-3) GF22 100 ugmRampD AB-233-NA 1 mgvialBiogenex AR-359-5ROncogene Sciences PC16 100 ug

FibronectinRampD RDI- bronabm

Heparan sulfateSeikagaku Amer Cat 370255 02 mg

CollagenCaltag rabbit anti pig collagen I amp III PS067 1 mlVector collagen Type I NCL-COLL-Ip 1ml

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Preparation and labeling of parafn sections

SIS was xed for 24 h with 10 buffered formalin The xed pieces about25 mm2 in size were processed and embedded in paraf n on edge and cut into5 sup1m thickness sections that were deparaf nized hydrated and labeled with theBioGenex Optimax automatic stainer A more extensive deparaf nization than isusual was employed because we have found more extensive dewaxing decreasesnonspeci c background uorescence in tissue sections Sections were dewaxed for3 min each in three changes of xylene Each week the rst solution is discardedan a fresh third solution is prepared The sections are then washed sequentiallyin two solutions of absolute ethanol then 95 75 and 50 for 5 min followed bytwo washes against deionized water For immunohistochemical labeling the ZymedST5050 AEC detection system was used A counterstain of hematoxylin generallywas employed to visualize nuclei and tissue architecture For uorescence labelingthe tyramide signal ampli cation system (TSA NEN Inc) was used to enhance thesignal to noise ratio with uorescence Nuclei were labeled with 10 sup1M Hoechst33258 in 25 ethanol-MOPSO buffer According to literature published by NENthis method generally achieves a roughly 100-fold enhancement of the speci c uorescence signal with no increase in background

Standardization of uorescence

Originally the plan to standardize uorescence was to use collagen as a standardwith any additional components being measured with a second color Collagenis present in all SIS and a pixel-by-pixel standardization of any component tocollagen could provide a structural map However as we show below thethickness of sections necessary to prevent shredding of the SIS precluded accuratequanti cation We therefore used an alternate approach in which pig intestinewas used as an arbitrary standard against which various SIS preparations couldbe compared Images were captured using a Nikon Microphot FXA microscopeequipped with a highly linear fast Hammamatsu Color Chilled 3CCD camerathat permitted image capture before signi cant fading of uorescence occurredQuanti cation was carried out using a separate system originally developed forcytology preparations [11] Ten elds were measured for each preparation afterthresholding to remove as much of the nonspeci c signal as possible

Antigen retrieval

A number of antigen retrieval approaches were investigated to determine if theyenhanced the signal Signal enhancement might result if the target protein is boundin a tight complex that masks the epitope recognized by the antibody or is maskedby formaldehyde cross-linking Antigen retrieval is the general process by whichselective denaturation reveals masked protein antigens to the antibody TargetingUnmasking Fluid (Signet) Citra Citra Plus and AR10 (BioGenex) are products

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Molecular mapping of SIS 1271

for denaturing supramolecular complexes in tissue sections and these were used todetermine if signal could be enhanced

RESULTS

5 vs 20 sup1m sections and sectioned formaldehyde-preserved tissue blocksvs frozen sections

The general standard for histopathologic examination is the 3 or 5 sup1m section ofparaf n-embedded formaldehyde-preserved material Unless the target is highlyexpressed the paraf n section usually is unsuitable for uorescence applicationsbecause of background uorescence Consequently uorescence assays on histo-logic material typically are performed on frozen sections The material to be exam-ined is frozen at dry ice or liquid nitrogen temperatures into a block of supportivematerial which then is sectioned with a microtome For various reasons frozensections do not retain morphology nearly so well as paraf n sections displayingmore distortions from the cutting process and often displaying something knownas lsquofreeze artifactrsquo Thus the examination of tissue material very much representsachieving a balance among competing limiting factors

These competitive forces are particularly relevant in examining SIS which istough and tends to tear or shred when sectioned into 5 sup1m frozen sections Thetearing tends to destroy anatomic relationships Sections cut at 20 sup1m retainmorphology but washing out unbound reagents becomes problematic Thus carefulattention must be paid to details of speci city To assist with determining speci citya sample of pig intestine was used to evaluate antibody speci city and the adequacyof the assay because the presence of distinct epithelium submucosa and muscularisallows evaluation of speci city Figure 1A illustrates dual labeling for collagen and bronectin in a 5-sup1m section of SIS Areas that are green express heparan sulfateAreas that are red express collagen and areas that are brown express both Thisis one of the best preserved 5 sup1m sections A more usual section is shown inFig 1B which shows heparan sulfate and collagen in SIS labeled with different uorophores Although the dual labels are clearly evident the structure of the SIShas essentially been lost

Figure 1C illustrates the speci city of labeling as assessed with a sample of intactpig intestine triply labeled for DNA (blue) collagen (red) and bronectin (green)The DNA label unambiguously identi es the villous epithelium The bronectinis seen mainly in the extracellular matrix of the epithelium while the collagen isfound mainly in the submucosa This distribution illustrates the speci city of theantibodies and shows that nonspeci c binding is minimal However with the usualshredding seen in Fig 1B quantitative analysis of SIS proved dif cult

With 20-sup1m frozen sections the morphology was preserved but the resultsbecame inconsistent The limiting factor seemed to be washing out of unboundreagents Systematic investigations showed that increasing the salt concentration in

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Figure 1 Photographs of labeled SIS and pig intestine All photomicrographs are at the samemagni cation indicated by the bar in N (A) Double uorescent label of bronectin (green) andcollagen (red) in a 5-sup1m frozen SIS section demonstrating occasional preservation of morphology(B) A more usual result showing shredding and loss of morphology in a 5-sup1m frozen SIS sectionlabeled for heparan sulfate proteoglycan (green) and collagen (red) (C) Triple label of pig intestinedemonstrating speci city of labeling for bronectin (green) collagen (red) and DNA (blue) Thegreen area on the right is the villous epithelium The brown area is the submucosa from which SISis obtained (D) Labeling of collagen in a 20 sup1m section of SIS by indirect assay (E)ndash(H) Se-quential sections of pig intestine (E F) or SIS (G H) labeled for collagen by TSA ampli cation(E G) and heparan sulfate using a conventional sandwich method (F H) with 5 sup1m sections ofparaf n-preserved blocks Note the lack of background in areas where collagen or heparan sulfateis absent even with the heparan sulfate assay which does not employ the TSA ampli cation Theboundary between the submucosa and epithelium is at the bottom of the picture (E G) (I) and (J)Immunohistochemicallabelingof pig intestine(I) and SIS (J) for heparan sulfate proteoglycanin 5 sup1mparaf n block sections (K) and (L) FGF2 labelingby uorescence(K) and immunohistochemistry(L)demonstrating diffuse brous labeling Note FGF2 seems to be concentrated on the lumenal surface(top L) (M) and (N) VEGF labeling in pig intestine (M) and SIS (N) demonstrating localization tovessels and loss from SIS

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Molecular mapping of SIS 1273

Figure 2 Titration of rabbit anticollagen (f) (positive) and (F)(negative control) in 20 sup1m frozensections of SIS using indirect immuno uorescence (sandwich) assay in comparison to TSA methodwith 5 sup1m sections The negative control substituted an unrelated serum at the same dilution as theprimary antibody The x-axis is arbitrary because the concentration of the polyclonal antibody wasunknown The difference between the positive and negative is proportional to the content of collagenin the sample The attening of the curve indicates saturation of binding sites and proportionality(s) shows the titration of the antibody using the TSA method on SIS and ( u ) on pig intestine Thedifferencebetween the two curves represents the difference in collagen immunoreactivitybetween thetwo specimens

wash buffers as well as the number of washes reduced the background uorescencebut never eliminated it In spite of these improvements consistent low backgroundcould not be obtained with the 20-sup1m sections Often speci c binding could beseen as bright areas against a low-intensity background These problems were ofleast signi cance for collagen which is highly abundant but were more signi cantfor markers that were of low abundance

Figure 2 illustrates the titration of the anti-collagen antibody with a conventionallsquosandwichrsquo assay on a 20-sup1m section of porcine intestine provided by Cook Biotechas being representative of the material from which SIS is made In the absenceof primary antibody there clearly is a background uorescence as shown bythe intercept on the y-axis The amount is the same with an unrelated primaryindicating that the background is due to failure to wash out all the uorophoreIncreasing the concentration of antibody yields the expected saturation of sites Inthe at portion of the curve the difference between background and the antibody-treated sample is directly proportional to the amount of target in this case collagen

Tyramide signal ampli cation (TSA) method

This technique offers a means to amplify the speci c signal arising from binding ofan individual antibody to its antigen in 5-sup1m paraf n sections thereby enhancingthe signal to noise ratio suf ciently to swamp out the nonspeci c uorescenceseen in paraf n sections This nonspeci c uorescence is not due to the antibody

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1274 R E Hurst and R B Bonner

because it appears in the absence of any reagents Instead of a uorochrome-labeledprimary or secondary the detection system consists of horseradish peroxidase(HRP) conjugated to the secondary antibody which then catalyzes deposition ofinsoluble biotin or dye-labeled tyramide in the immediate vicinity of the HRPAs each HRP moiety can generate numerous tyramide moieties a marked signalenhancement is observed The titration of antibody in the TSA system also isshown in Fig 2 The nonspeci c uorescence background is virtually undetectablein the controls for these assays of collagen which is a high-expression proteinand represents the ideal case for both conventional immuno uorescence assays andthe TSA ampli cation method The uorescence increases with primary antibodyconcentration from essentially zero indicating the ampli cation of the speci csignal over the nonspeci c signal As with the common lsquosandwichrsquo method thecurve breaks over indicating saturation of sites The difference in uorescenceintensity between the SIS and pig samples is proportional to the difference incollagen I content of the two samples

Heparan sulfate proteoglycans are of interest because they bind growth peptides invivo [18] The HS-PG is clearly detectable in the SIS material without ampli cation(Fig 1B F and H) Figure 3 shows the titration of heparan sulfate with theTSA method and demonstrates the presence of HS-PG in both the submucosalregion of the pig intestine and the SIS with the intestinal sample having a higherconcentration of the proteoglycan Figure 1EndashH shows the appearance of thecollagen enhanced by the TSA method and HS-PG labeled with a conventionallsquosandwichrsquo assay Collagen is labeled with red while heparan sulfate proteoglycanis labeled in green Figures 1E and F demonstrate the intact pig intestine whileFig 1G and H demonstrate SIS Note that the sections seem to be cut along differentplanes The glands and vessels are positive for heparan sulfate with very little

Figure 3 Titration of anti-heparan sulfate antibody using TSA method for SIS (s) and pigintestine (u )

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Molecular mapping of SIS 1275

background particularly in the SIS (Fig 1G and H) These high abundance proteinscould be quanti ed either from conventional immun uorescence even on paraf nsections or with the TSA method

With the TSA method negative controls are undetectable for measurement asare the lowest concentrations of this titration curve (Fig 3) Binding appears tobe very speci c and intense Both the unprocessed porcine intestine and the SISmaterial demonstrate speci c binding of anti-heparan sulfate antibody Vessels arethe primary positive component in the submucosa The values indicate that there issome loss of antigenicity (about 35) in SIS material compared to the intestinesample but clearly heparan sulfate is present within the body of the SIS itself(Fig 1H) The uorescence data are con rmed by immunohistochemical labelingwhich is demonstrated in Fig 1I and J Clearly the heparan sulfate proteoglycan ismore prevalent in the section derived from the pig than from the SIS This con rmsthe loss of some of the heparan sulfate proteoglycans during the processing

Growth peptides and low abundance components of SIS

Clearly the TSA procedure can identify and quantify components in tissues andSIS We next sought to apply it to components likely to be present in lowerconcentrations such as growth peptides which might not be evident against thebackground uorescence The distribution of FGF2 TGFmacr and VEGF in SIS wereinvestigated in relation to the amounts seen in pig intestine

Figure 4 illustrates a titration of the FGF2 antibody in the TSA system Thetwo points shown with the circle and square represent the controls which representan unrelated antibody of the same isotype as the primary used at the highestconcentration the speci c primary is used The lowest background values of thirdygray level values is still well below the speci c signal The values on the ordinate arearbitrary and are adjusted to keep the signal within the linear range of the measuring

Figure 4 Titration of anti-FGF2 antibody using TSA method for SIS (s) and pig intestine (u )(fF) represent an unrelated isotype-controlledmonoclonal antibody used as a negative control at thehighest concentrationused in the titration of the speci c antibody

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1276 R E Hurst and R B Bonner

Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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Molecular mapping of SIS 1269

blocks was then used to compare the amounts of several growth peptides and matrixmolecules in SIS in comparison to pig mucosa

MATERIALS AND METHODS

SIS was obtained from Cook Biotech (West Lafayette IN USA) The antibodiesused in labeling are described in Table 1

Preparation and labeling of frozen sections

Approximate 25 mm2 samples of SIS were positioned on edge in freezing cassettesand surrounded by OCT compound The cassettes were then frozen in isopentanecooled with liquid nitrogen The frozen material was then sectioned on a cryostaticmicrotome at either 5 or 20 sup1m thickness Each preparation was examined byhematoxylin and eosin (HampE) staining to ensure the proper orientation of theSIS Sections were captured on slides and labeled on an automatic stainer theBioGenex Optimax The slides were incubated with the proper primary antibodywashed and then incubated with a secondary biotinylated antibody The tertiaryreagent consisted of Alexa-488 or Texas Red-labeled avidin With proper use ofisotype-speci c secondary antibodies and monoclonal primaries double-labelingwas possible

Table 1Antibodies used in study

TGFmacr1RampD MAB1835 500 microgramsvialOncogene Sciences GF33L 100 ugOncogene Sciences PF052 1 ug

VEGFRampD MAB293 500 microgramsvialBiogenex AR360-5R

Basic FGFOncogene Sciences (AB-3) GF22 100 ugmRampD AB-233-NA 1 mgvialBiogenex AR-359-5ROncogene Sciences PC16 100 ug

FibronectinRampD RDI- bronabm

Heparan sulfateSeikagaku Amer Cat 370255 02 mg

CollagenCaltag rabbit anti pig collagen I amp III PS067 1 mlVector collagen Type I NCL-COLL-Ip 1ml

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1270 R E Hurst and R B Bonner

Preparation and labeling of parafn sections

SIS was xed for 24 h with 10 buffered formalin The xed pieces about25 mm2 in size were processed and embedded in paraf n on edge and cut into5 sup1m thickness sections that were deparaf nized hydrated and labeled with theBioGenex Optimax automatic stainer A more extensive deparaf nization than isusual was employed because we have found more extensive dewaxing decreasesnonspeci c background uorescence in tissue sections Sections were dewaxed for3 min each in three changes of xylene Each week the rst solution is discardedan a fresh third solution is prepared The sections are then washed sequentiallyin two solutions of absolute ethanol then 95 75 and 50 for 5 min followed bytwo washes against deionized water For immunohistochemical labeling the ZymedST5050 AEC detection system was used A counterstain of hematoxylin generallywas employed to visualize nuclei and tissue architecture For uorescence labelingthe tyramide signal ampli cation system (TSA NEN Inc) was used to enhance thesignal to noise ratio with uorescence Nuclei were labeled with 10 sup1M Hoechst33258 in 25 ethanol-MOPSO buffer According to literature published by NENthis method generally achieves a roughly 100-fold enhancement of the speci c uorescence signal with no increase in background

Standardization of uorescence

Originally the plan to standardize uorescence was to use collagen as a standardwith any additional components being measured with a second color Collagenis present in all SIS and a pixel-by-pixel standardization of any component tocollagen could provide a structural map However as we show below thethickness of sections necessary to prevent shredding of the SIS precluded accuratequanti cation We therefore used an alternate approach in which pig intestinewas used as an arbitrary standard against which various SIS preparations couldbe compared Images were captured using a Nikon Microphot FXA microscopeequipped with a highly linear fast Hammamatsu Color Chilled 3CCD camerathat permitted image capture before signi cant fading of uorescence occurredQuanti cation was carried out using a separate system originally developed forcytology preparations [11] Ten elds were measured for each preparation afterthresholding to remove as much of the nonspeci c signal as possible

Antigen retrieval

A number of antigen retrieval approaches were investigated to determine if theyenhanced the signal Signal enhancement might result if the target protein is boundin a tight complex that masks the epitope recognized by the antibody or is maskedby formaldehyde cross-linking Antigen retrieval is the general process by whichselective denaturation reveals masked protein antigens to the antibody TargetingUnmasking Fluid (Signet) Citra Citra Plus and AR10 (BioGenex) are products

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Molecular mapping of SIS 1271

for denaturing supramolecular complexes in tissue sections and these were used todetermine if signal could be enhanced

RESULTS

5 vs 20 sup1m sections and sectioned formaldehyde-preserved tissue blocksvs frozen sections

The general standard for histopathologic examination is the 3 or 5 sup1m section ofparaf n-embedded formaldehyde-preserved material Unless the target is highlyexpressed the paraf n section usually is unsuitable for uorescence applicationsbecause of background uorescence Consequently uorescence assays on histo-logic material typically are performed on frozen sections The material to be exam-ined is frozen at dry ice or liquid nitrogen temperatures into a block of supportivematerial which then is sectioned with a microtome For various reasons frozensections do not retain morphology nearly so well as paraf n sections displayingmore distortions from the cutting process and often displaying something knownas lsquofreeze artifactrsquo Thus the examination of tissue material very much representsachieving a balance among competing limiting factors

These competitive forces are particularly relevant in examining SIS which istough and tends to tear or shred when sectioned into 5 sup1m frozen sections Thetearing tends to destroy anatomic relationships Sections cut at 20 sup1m retainmorphology but washing out unbound reagents becomes problematic Thus carefulattention must be paid to details of speci city To assist with determining speci citya sample of pig intestine was used to evaluate antibody speci city and the adequacyof the assay because the presence of distinct epithelium submucosa and muscularisallows evaluation of speci city Figure 1A illustrates dual labeling for collagen and bronectin in a 5-sup1m section of SIS Areas that are green express heparan sulfateAreas that are red express collagen and areas that are brown express both Thisis one of the best preserved 5 sup1m sections A more usual section is shown inFig 1B which shows heparan sulfate and collagen in SIS labeled with different uorophores Although the dual labels are clearly evident the structure of the SIShas essentially been lost

Figure 1C illustrates the speci city of labeling as assessed with a sample of intactpig intestine triply labeled for DNA (blue) collagen (red) and bronectin (green)The DNA label unambiguously identi es the villous epithelium The bronectinis seen mainly in the extracellular matrix of the epithelium while the collagen isfound mainly in the submucosa This distribution illustrates the speci city of theantibodies and shows that nonspeci c binding is minimal However with the usualshredding seen in Fig 1B quantitative analysis of SIS proved dif cult

With 20-sup1m frozen sections the morphology was preserved but the resultsbecame inconsistent The limiting factor seemed to be washing out of unboundreagents Systematic investigations showed that increasing the salt concentration in

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1272 R E Hurst and R B Bonner

Figure 1 Photographs of labeled SIS and pig intestine All photomicrographs are at the samemagni cation indicated by the bar in N (A) Double uorescent label of bronectin (green) andcollagen (red) in a 5-sup1m frozen SIS section demonstrating occasional preservation of morphology(B) A more usual result showing shredding and loss of morphology in a 5-sup1m frozen SIS sectionlabeled for heparan sulfate proteoglycan (green) and collagen (red) (C) Triple label of pig intestinedemonstrating speci city of labeling for bronectin (green) collagen (red) and DNA (blue) Thegreen area on the right is the villous epithelium The brown area is the submucosa from which SISis obtained (D) Labeling of collagen in a 20 sup1m section of SIS by indirect assay (E)ndash(H) Se-quential sections of pig intestine (E F) or SIS (G H) labeled for collagen by TSA ampli cation(E G) and heparan sulfate using a conventional sandwich method (F H) with 5 sup1m sections ofparaf n-preserved blocks Note the lack of background in areas where collagen or heparan sulfateis absent even with the heparan sulfate assay which does not employ the TSA ampli cation Theboundary between the submucosa and epithelium is at the bottom of the picture (E G) (I) and (J)Immunohistochemicallabelingof pig intestine(I) and SIS (J) for heparan sulfate proteoglycanin 5 sup1mparaf n block sections (K) and (L) FGF2 labelingby uorescence(K) and immunohistochemistry(L)demonstrating diffuse brous labeling Note FGF2 seems to be concentrated on the lumenal surface(top L) (M) and (N) VEGF labeling in pig intestine (M) and SIS (N) demonstrating localization tovessels and loss from SIS

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Molecular mapping of SIS 1273

Figure 2 Titration of rabbit anticollagen (f) (positive) and (F)(negative control) in 20 sup1m frozensections of SIS using indirect immuno uorescence (sandwich) assay in comparison to TSA methodwith 5 sup1m sections The negative control substituted an unrelated serum at the same dilution as theprimary antibody The x-axis is arbitrary because the concentration of the polyclonal antibody wasunknown The difference between the positive and negative is proportional to the content of collagenin the sample The attening of the curve indicates saturation of binding sites and proportionality(s) shows the titration of the antibody using the TSA method on SIS and ( u ) on pig intestine Thedifferencebetween the two curves represents the difference in collagen immunoreactivitybetween thetwo specimens

wash buffers as well as the number of washes reduced the background uorescencebut never eliminated it In spite of these improvements consistent low backgroundcould not be obtained with the 20-sup1m sections Often speci c binding could beseen as bright areas against a low-intensity background These problems were ofleast signi cance for collagen which is highly abundant but were more signi cantfor markers that were of low abundance

Figure 2 illustrates the titration of the anti-collagen antibody with a conventionallsquosandwichrsquo assay on a 20-sup1m section of porcine intestine provided by Cook Biotechas being representative of the material from which SIS is made In the absenceof primary antibody there clearly is a background uorescence as shown bythe intercept on the y-axis The amount is the same with an unrelated primaryindicating that the background is due to failure to wash out all the uorophoreIncreasing the concentration of antibody yields the expected saturation of sites Inthe at portion of the curve the difference between background and the antibody-treated sample is directly proportional to the amount of target in this case collagen

Tyramide signal ampli cation (TSA) method

This technique offers a means to amplify the speci c signal arising from binding ofan individual antibody to its antigen in 5-sup1m paraf n sections thereby enhancingthe signal to noise ratio suf ciently to swamp out the nonspeci c uorescenceseen in paraf n sections This nonspeci c uorescence is not due to the antibody

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1274 R E Hurst and R B Bonner

because it appears in the absence of any reagents Instead of a uorochrome-labeledprimary or secondary the detection system consists of horseradish peroxidase(HRP) conjugated to the secondary antibody which then catalyzes deposition ofinsoluble biotin or dye-labeled tyramide in the immediate vicinity of the HRPAs each HRP moiety can generate numerous tyramide moieties a marked signalenhancement is observed The titration of antibody in the TSA system also isshown in Fig 2 The nonspeci c uorescence background is virtually undetectablein the controls for these assays of collagen which is a high-expression proteinand represents the ideal case for both conventional immuno uorescence assays andthe TSA ampli cation method The uorescence increases with primary antibodyconcentration from essentially zero indicating the ampli cation of the speci csignal over the nonspeci c signal As with the common lsquosandwichrsquo method thecurve breaks over indicating saturation of sites The difference in uorescenceintensity between the SIS and pig samples is proportional to the difference incollagen I content of the two samples

Heparan sulfate proteoglycans are of interest because they bind growth peptides invivo [18] The HS-PG is clearly detectable in the SIS material without ampli cation(Fig 1B F and H) Figure 3 shows the titration of heparan sulfate with theTSA method and demonstrates the presence of HS-PG in both the submucosalregion of the pig intestine and the SIS with the intestinal sample having a higherconcentration of the proteoglycan Figure 1EndashH shows the appearance of thecollagen enhanced by the TSA method and HS-PG labeled with a conventionallsquosandwichrsquo assay Collagen is labeled with red while heparan sulfate proteoglycanis labeled in green Figures 1E and F demonstrate the intact pig intestine whileFig 1G and H demonstrate SIS Note that the sections seem to be cut along differentplanes The glands and vessels are positive for heparan sulfate with very little

Figure 3 Titration of anti-heparan sulfate antibody using TSA method for SIS (s) and pigintestine (u )

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Molecular mapping of SIS 1275

background particularly in the SIS (Fig 1G and H) These high abundance proteinscould be quanti ed either from conventional immun uorescence even on paraf nsections or with the TSA method

With the TSA method negative controls are undetectable for measurement asare the lowest concentrations of this titration curve (Fig 3) Binding appears tobe very speci c and intense Both the unprocessed porcine intestine and the SISmaterial demonstrate speci c binding of anti-heparan sulfate antibody Vessels arethe primary positive component in the submucosa The values indicate that there issome loss of antigenicity (about 35) in SIS material compared to the intestinesample but clearly heparan sulfate is present within the body of the SIS itself(Fig 1H) The uorescence data are con rmed by immunohistochemical labelingwhich is demonstrated in Fig 1I and J Clearly the heparan sulfate proteoglycan ismore prevalent in the section derived from the pig than from the SIS This con rmsthe loss of some of the heparan sulfate proteoglycans during the processing

Growth peptides and low abundance components of SIS

Clearly the TSA procedure can identify and quantify components in tissues andSIS We next sought to apply it to components likely to be present in lowerconcentrations such as growth peptides which might not be evident against thebackground uorescence The distribution of FGF2 TGFmacr and VEGF in SIS wereinvestigated in relation to the amounts seen in pig intestine

Figure 4 illustrates a titration of the FGF2 antibody in the TSA system Thetwo points shown with the circle and square represent the controls which representan unrelated antibody of the same isotype as the primary used at the highestconcentration the speci c primary is used The lowest background values of thirdygray level values is still well below the speci c signal The values on the ordinate arearbitrary and are adjusted to keep the signal within the linear range of the measuring

Figure 4 Titration of anti-FGF2 antibody using TSA method for SIS (s) and pig intestine (u )(fF) represent an unrelated isotype-controlledmonoclonal antibody used as a negative control at thehighest concentrationused in the titration of the speci c antibody

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1276 R E Hurst and R B Bonner

Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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1270 R E Hurst and R B Bonner

Preparation and labeling of parafn sections

SIS was xed for 24 h with 10 buffered formalin The xed pieces about25 mm2 in size were processed and embedded in paraf n on edge and cut into5 sup1m thickness sections that were deparaf nized hydrated and labeled with theBioGenex Optimax automatic stainer A more extensive deparaf nization than isusual was employed because we have found more extensive dewaxing decreasesnonspeci c background uorescence in tissue sections Sections were dewaxed for3 min each in three changes of xylene Each week the rst solution is discardedan a fresh third solution is prepared The sections are then washed sequentiallyin two solutions of absolute ethanol then 95 75 and 50 for 5 min followed bytwo washes against deionized water For immunohistochemical labeling the ZymedST5050 AEC detection system was used A counterstain of hematoxylin generallywas employed to visualize nuclei and tissue architecture For uorescence labelingthe tyramide signal ampli cation system (TSA NEN Inc) was used to enhance thesignal to noise ratio with uorescence Nuclei were labeled with 10 sup1M Hoechst33258 in 25 ethanol-MOPSO buffer According to literature published by NENthis method generally achieves a roughly 100-fold enhancement of the speci c uorescence signal with no increase in background

Standardization of uorescence

Originally the plan to standardize uorescence was to use collagen as a standardwith any additional components being measured with a second color Collagenis present in all SIS and a pixel-by-pixel standardization of any component tocollagen could provide a structural map However as we show below thethickness of sections necessary to prevent shredding of the SIS precluded accuratequanti cation We therefore used an alternate approach in which pig intestinewas used as an arbitrary standard against which various SIS preparations couldbe compared Images were captured using a Nikon Microphot FXA microscopeequipped with a highly linear fast Hammamatsu Color Chilled 3CCD camerathat permitted image capture before signi cant fading of uorescence occurredQuanti cation was carried out using a separate system originally developed forcytology preparations [11] Ten elds were measured for each preparation afterthresholding to remove as much of the nonspeci c signal as possible

Antigen retrieval

A number of antigen retrieval approaches were investigated to determine if theyenhanced the signal Signal enhancement might result if the target protein is boundin a tight complex that masks the epitope recognized by the antibody or is maskedby formaldehyde cross-linking Antigen retrieval is the general process by whichselective denaturation reveals masked protein antigens to the antibody TargetingUnmasking Fluid (Signet) Citra Citra Plus and AR10 (BioGenex) are products

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Molecular mapping of SIS 1271

for denaturing supramolecular complexes in tissue sections and these were used todetermine if signal could be enhanced

RESULTS

5 vs 20 sup1m sections and sectioned formaldehyde-preserved tissue blocksvs frozen sections

The general standard for histopathologic examination is the 3 or 5 sup1m section ofparaf n-embedded formaldehyde-preserved material Unless the target is highlyexpressed the paraf n section usually is unsuitable for uorescence applicationsbecause of background uorescence Consequently uorescence assays on histo-logic material typically are performed on frozen sections The material to be exam-ined is frozen at dry ice or liquid nitrogen temperatures into a block of supportivematerial which then is sectioned with a microtome For various reasons frozensections do not retain morphology nearly so well as paraf n sections displayingmore distortions from the cutting process and often displaying something knownas lsquofreeze artifactrsquo Thus the examination of tissue material very much representsachieving a balance among competing limiting factors

These competitive forces are particularly relevant in examining SIS which istough and tends to tear or shred when sectioned into 5 sup1m frozen sections Thetearing tends to destroy anatomic relationships Sections cut at 20 sup1m retainmorphology but washing out unbound reagents becomes problematic Thus carefulattention must be paid to details of speci city To assist with determining speci citya sample of pig intestine was used to evaluate antibody speci city and the adequacyof the assay because the presence of distinct epithelium submucosa and muscularisallows evaluation of speci city Figure 1A illustrates dual labeling for collagen and bronectin in a 5-sup1m section of SIS Areas that are green express heparan sulfateAreas that are red express collagen and areas that are brown express both Thisis one of the best preserved 5 sup1m sections A more usual section is shown inFig 1B which shows heparan sulfate and collagen in SIS labeled with different uorophores Although the dual labels are clearly evident the structure of the SIShas essentially been lost

Figure 1C illustrates the speci city of labeling as assessed with a sample of intactpig intestine triply labeled for DNA (blue) collagen (red) and bronectin (green)The DNA label unambiguously identi es the villous epithelium The bronectinis seen mainly in the extracellular matrix of the epithelium while the collagen isfound mainly in the submucosa This distribution illustrates the speci city of theantibodies and shows that nonspeci c binding is minimal However with the usualshredding seen in Fig 1B quantitative analysis of SIS proved dif cult

With 20-sup1m frozen sections the morphology was preserved but the resultsbecame inconsistent The limiting factor seemed to be washing out of unboundreagents Systematic investigations showed that increasing the salt concentration in

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1272 R E Hurst and R B Bonner

Figure 1 Photographs of labeled SIS and pig intestine All photomicrographs are at the samemagni cation indicated by the bar in N (A) Double uorescent label of bronectin (green) andcollagen (red) in a 5-sup1m frozen SIS section demonstrating occasional preservation of morphology(B) A more usual result showing shredding and loss of morphology in a 5-sup1m frozen SIS sectionlabeled for heparan sulfate proteoglycan (green) and collagen (red) (C) Triple label of pig intestinedemonstrating speci city of labeling for bronectin (green) collagen (red) and DNA (blue) Thegreen area on the right is the villous epithelium The brown area is the submucosa from which SISis obtained (D) Labeling of collagen in a 20 sup1m section of SIS by indirect assay (E)ndash(H) Se-quential sections of pig intestine (E F) or SIS (G H) labeled for collagen by TSA ampli cation(E G) and heparan sulfate using a conventional sandwich method (F H) with 5 sup1m sections ofparaf n-preserved blocks Note the lack of background in areas where collagen or heparan sulfateis absent even with the heparan sulfate assay which does not employ the TSA ampli cation Theboundary between the submucosa and epithelium is at the bottom of the picture (E G) (I) and (J)Immunohistochemicallabelingof pig intestine(I) and SIS (J) for heparan sulfate proteoglycanin 5 sup1mparaf n block sections (K) and (L) FGF2 labelingby uorescence(K) and immunohistochemistry(L)demonstrating diffuse brous labeling Note FGF2 seems to be concentrated on the lumenal surface(top L) (M) and (N) VEGF labeling in pig intestine (M) and SIS (N) demonstrating localization tovessels and loss from SIS

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Molecular mapping of SIS 1273

Figure 2 Titration of rabbit anticollagen (f) (positive) and (F)(negative control) in 20 sup1m frozensections of SIS using indirect immuno uorescence (sandwich) assay in comparison to TSA methodwith 5 sup1m sections The negative control substituted an unrelated serum at the same dilution as theprimary antibody The x-axis is arbitrary because the concentration of the polyclonal antibody wasunknown The difference between the positive and negative is proportional to the content of collagenin the sample The attening of the curve indicates saturation of binding sites and proportionality(s) shows the titration of the antibody using the TSA method on SIS and ( u ) on pig intestine Thedifferencebetween the two curves represents the difference in collagen immunoreactivitybetween thetwo specimens

wash buffers as well as the number of washes reduced the background uorescencebut never eliminated it In spite of these improvements consistent low backgroundcould not be obtained with the 20-sup1m sections Often speci c binding could beseen as bright areas against a low-intensity background These problems were ofleast signi cance for collagen which is highly abundant but were more signi cantfor markers that were of low abundance

Figure 2 illustrates the titration of the anti-collagen antibody with a conventionallsquosandwichrsquo assay on a 20-sup1m section of porcine intestine provided by Cook Biotechas being representative of the material from which SIS is made In the absenceof primary antibody there clearly is a background uorescence as shown bythe intercept on the y-axis The amount is the same with an unrelated primaryindicating that the background is due to failure to wash out all the uorophoreIncreasing the concentration of antibody yields the expected saturation of sites Inthe at portion of the curve the difference between background and the antibody-treated sample is directly proportional to the amount of target in this case collagen

Tyramide signal ampli cation (TSA) method

This technique offers a means to amplify the speci c signal arising from binding ofan individual antibody to its antigen in 5-sup1m paraf n sections thereby enhancingthe signal to noise ratio suf ciently to swamp out the nonspeci c uorescenceseen in paraf n sections This nonspeci c uorescence is not due to the antibody

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1274 R E Hurst and R B Bonner

because it appears in the absence of any reagents Instead of a uorochrome-labeledprimary or secondary the detection system consists of horseradish peroxidase(HRP) conjugated to the secondary antibody which then catalyzes deposition ofinsoluble biotin or dye-labeled tyramide in the immediate vicinity of the HRPAs each HRP moiety can generate numerous tyramide moieties a marked signalenhancement is observed The titration of antibody in the TSA system also isshown in Fig 2 The nonspeci c uorescence background is virtually undetectablein the controls for these assays of collagen which is a high-expression proteinand represents the ideal case for both conventional immuno uorescence assays andthe TSA ampli cation method The uorescence increases with primary antibodyconcentration from essentially zero indicating the ampli cation of the speci csignal over the nonspeci c signal As with the common lsquosandwichrsquo method thecurve breaks over indicating saturation of sites The difference in uorescenceintensity between the SIS and pig samples is proportional to the difference incollagen I content of the two samples

Heparan sulfate proteoglycans are of interest because they bind growth peptides invivo [18] The HS-PG is clearly detectable in the SIS material without ampli cation(Fig 1B F and H) Figure 3 shows the titration of heparan sulfate with theTSA method and demonstrates the presence of HS-PG in both the submucosalregion of the pig intestine and the SIS with the intestinal sample having a higherconcentration of the proteoglycan Figure 1EndashH shows the appearance of thecollagen enhanced by the TSA method and HS-PG labeled with a conventionallsquosandwichrsquo assay Collagen is labeled with red while heparan sulfate proteoglycanis labeled in green Figures 1E and F demonstrate the intact pig intestine whileFig 1G and H demonstrate SIS Note that the sections seem to be cut along differentplanes The glands and vessels are positive for heparan sulfate with very little

Figure 3 Titration of anti-heparan sulfate antibody using TSA method for SIS (s) and pigintestine (u )

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Molecular mapping of SIS 1275

background particularly in the SIS (Fig 1G and H) These high abundance proteinscould be quanti ed either from conventional immun uorescence even on paraf nsections or with the TSA method

With the TSA method negative controls are undetectable for measurement asare the lowest concentrations of this titration curve (Fig 3) Binding appears tobe very speci c and intense Both the unprocessed porcine intestine and the SISmaterial demonstrate speci c binding of anti-heparan sulfate antibody Vessels arethe primary positive component in the submucosa The values indicate that there issome loss of antigenicity (about 35) in SIS material compared to the intestinesample but clearly heparan sulfate is present within the body of the SIS itself(Fig 1H) The uorescence data are con rmed by immunohistochemical labelingwhich is demonstrated in Fig 1I and J Clearly the heparan sulfate proteoglycan ismore prevalent in the section derived from the pig than from the SIS This con rmsthe loss of some of the heparan sulfate proteoglycans during the processing

Growth peptides and low abundance components of SIS

Clearly the TSA procedure can identify and quantify components in tissues andSIS We next sought to apply it to components likely to be present in lowerconcentrations such as growth peptides which might not be evident against thebackground uorescence The distribution of FGF2 TGFmacr and VEGF in SIS wereinvestigated in relation to the amounts seen in pig intestine

Figure 4 illustrates a titration of the FGF2 antibody in the TSA system Thetwo points shown with the circle and square represent the controls which representan unrelated antibody of the same isotype as the primary used at the highestconcentration the speci c primary is used The lowest background values of thirdygray level values is still well below the speci c signal The values on the ordinate arearbitrary and are adjusted to keep the signal within the linear range of the measuring

Figure 4 Titration of anti-FGF2 antibody using TSA method for SIS (s) and pig intestine (u )(fF) represent an unrelated isotype-controlledmonoclonal antibody used as a negative control at thehighest concentrationused in the titration of the speci c antibody

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1276 R E Hurst and R B Bonner

Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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for denaturing supramolecular complexes in tissue sections and these were used todetermine if signal could be enhanced

RESULTS

5 vs 20 sup1m sections and sectioned formaldehyde-preserved tissue blocksvs frozen sections

The general standard for histopathologic examination is the 3 or 5 sup1m section ofparaf n-embedded formaldehyde-preserved material Unless the target is highlyexpressed the paraf n section usually is unsuitable for uorescence applicationsbecause of background uorescence Consequently uorescence assays on histo-logic material typically are performed on frozen sections The material to be exam-ined is frozen at dry ice or liquid nitrogen temperatures into a block of supportivematerial which then is sectioned with a microtome For various reasons frozensections do not retain morphology nearly so well as paraf n sections displayingmore distortions from the cutting process and often displaying something knownas lsquofreeze artifactrsquo Thus the examination of tissue material very much representsachieving a balance among competing limiting factors

These competitive forces are particularly relevant in examining SIS which istough and tends to tear or shred when sectioned into 5 sup1m frozen sections Thetearing tends to destroy anatomic relationships Sections cut at 20 sup1m retainmorphology but washing out unbound reagents becomes problematic Thus carefulattention must be paid to details of speci city To assist with determining speci citya sample of pig intestine was used to evaluate antibody speci city and the adequacyof the assay because the presence of distinct epithelium submucosa and muscularisallows evaluation of speci city Figure 1A illustrates dual labeling for collagen and bronectin in a 5-sup1m section of SIS Areas that are green express heparan sulfateAreas that are red express collagen and areas that are brown express both Thisis one of the best preserved 5 sup1m sections A more usual section is shown inFig 1B which shows heparan sulfate and collagen in SIS labeled with different uorophores Although the dual labels are clearly evident the structure of the SIShas essentially been lost

Figure 1C illustrates the speci city of labeling as assessed with a sample of intactpig intestine triply labeled for DNA (blue) collagen (red) and bronectin (green)The DNA label unambiguously identi es the villous epithelium The bronectinis seen mainly in the extracellular matrix of the epithelium while the collagen isfound mainly in the submucosa This distribution illustrates the speci city of theantibodies and shows that nonspeci c binding is minimal However with the usualshredding seen in Fig 1B quantitative analysis of SIS proved dif cult

With 20-sup1m frozen sections the morphology was preserved but the resultsbecame inconsistent The limiting factor seemed to be washing out of unboundreagents Systematic investigations showed that increasing the salt concentration in

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Figure 1 Photographs of labeled SIS and pig intestine All photomicrographs are at the samemagni cation indicated by the bar in N (A) Double uorescent label of bronectin (green) andcollagen (red) in a 5-sup1m frozen SIS section demonstrating occasional preservation of morphology(B) A more usual result showing shredding and loss of morphology in a 5-sup1m frozen SIS sectionlabeled for heparan sulfate proteoglycan (green) and collagen (red) (C) Triple label of pig intestinedemonstrating speci city of labeling for bronectin (green) collagen (red) and DNA (blue) Thegreen area on the right is the villous epithelium The brown area is the submucosa from which SISis obtained (D) Labeling of collagen in a 20 sup1m section of SIS by indirect assay (E)ndash(H) Se-quential sections of pig intestine (E F) or SIS (G H) labeled for collagen by TSA ampli cation(E G) and heparan sulfate using a conventional sandwich method (F H) with 5 sup1m sections ofparaf n-preserved blocks Note the lack of background in areas where collagen or heparan sulfateis absent even with the heparan sulfate assay which does not employ the TSA ampli cation Theboundary between the submucosa and epithelium is at the bottom of the picture (E G) (I) and (J)Immunohistochemicallabelingof pig intestine(I) and SIS (J) for heparan sulfate proteoglycanin 5 sup1mparaf n block sections (K) and (L) FGF2 labelingby uorescence(K) and immunohistochemistry(L)demonstrating diffuse brous labeling Note FGF2 seems to be concentrated on the lumenal surface(top L) (M) and (N) VEGF labeling in pig intestine (M) and SIS (N) demonstrating localization tovessels and loss from SIS

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Molecular mapping of SIS 1273

Figure 2 Titration of rabbit anticollagen (f) (positive) and (F)(negative control) in 20 sup1m frozensections of SIS using indirect immuno uorescence (sandwich) assay in comparison to TSA methodwith 5 sup1m sections The negative control substituted an unrelated serum at the same dilution as theprimary antibody The x-axis is arbitrary because the concentration of the polyclonal antibody wasunknown The difference between the positive and negative is proportional to the content of collagenin the sample The attening of the curve indicates saturation of binding sites and proportionality(s) shows the titration of the antibody using the TSA method on SIS and ( u ) on pig intestine Thedifferencebetween the two curves represents the difference in collagen immunoreactivitybetween thetwo specimens

wash buffers as well as the number of washes reduced the background uorescencebut never eliminated it In spite of these improvements consistent low backgroundcould not be obtained with the 20-sup1m sections Often speci c binding could beseen as bright areas against a low-intensity background These problems were ofleast signi cance for collagen which is highly abundant but were more signi cantfor markers that were of low abundance

Figure 2 illustrates the titration of the anti-collagen antibody with a conventionallsquosandwichrsquo assay on a 20-sup1m section of porcine intestine provided by Cook Biotechas being representative of the material from which SIS is made In the absenceof primary antibody there clearly is a background uorescence as shown bythe intercept on the y-axis The amount is the same with an unrelated primaryindicating that the background is due to failure to wash out all the uorophoreIncreasing the concentration of antibody yields the expected saturation of sites Inthe at portion of the curve the difference between background and the antibody-treated sample is directly proportional to the amount of target in this case collagen

Tyramide signal ampli cation (TSA) method

This technique offers a means to amplify the speci c signal arising from binding ofan individual antibody to its antigen in 5-sup1m paraf n sections thereby enhancingthe signal to noise ratio suf ciently to swamp out the nonspeci c uorescenceseen in paraf n sections This nonspeci c uorescence is not due to the antibody

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1274 R E Hurst and R B Bonner

because it appears in the absence of any reagents Instead of a uorochrome-labeledprimary or secondary the detection system consists of horseradish peroxidase(HRP) conjugated to the secondary antibody which then catalyzes deposition ofinsoluble biotin or dye-labeled tyramide in the immediate vicinity of the HRPAs each HRP moiety can generate numerous tyramide moieties a marked signalenhancement is observed The titration of antibody in the TSA system also isshown in Fig 2 The nonspeci c uorescence background is virtually undetectablein the controls for these assays of collagen which is a high-expression proteinand represents the ideal case for both conventional immuno uorescence assays andthe TSA ampli cation method The uorescence increases with primary antibodyconcentration from essentially zero indicating the ampli cation of the speci csignal over the nonspeci c signal As with the common lsquosandwichrsquo method thecurve breaks over indicating saturation of sites The difference in uorescenceintensity between the SIS and pig samples is proportional to the difference incollagen I content of the two samples

Heparan sulfate proteoglycans are of interest because they bind growth peptides invivo [18] The HS-PG is clearly detectable in the SIS material without ampli cation(Fig 1B F and H) Figure 3 shows the titration of heparan sulfate with theTSA method and demonstrates the presence of HS-PG in both the submucosalregion of the pig intestine and the SIS with the intestinal sample having a higherconcentration of the proteoglycan Figure 1EndashH shows the appearance of thecollagen enhanced by the TSA method and HS-PG labeled with a conventionallsquosandwichrsquo assay Collagen is labeled with red while heparan sulfate proteoglycanis labeled in green Figures 1E and F demonstrate the intact pig intestine whileFig 1G and H demonstrate SIS Note that the sections seem to be cut along differentplanes The glands and vessels are positive for heparan sulfate with very little

Figure 3 Titration of anti-heparan sulfate antibody using TSA method for SIS (s) and pigintestine (u )

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Molecular mapping of SIS 1275

background particularly in the SIS (Fig 1G and H) These high abundance proteinscould be quanti ed either from conventional immun uorescence even on paraf nsections or with the TSA method

With the TSA method negative controls are undetectable for measurement asare the lowest concentrations of this titration curve (Fig 3) Binding appears tobe very speci c and intense Both the unprocessed porcine intestine and the SISmaterial demonstrate speci c binding of anti-heparan sulfate antibody Vessels arethe primary positive component in the submucosa The values indicate that there issome loss of antigenicity (about 35) in SIS material compared to the intestinesample but clearly heparan sulfate is present within the body of the SIS itself(Fig 1H) The uorescence data are con rmed by immunohistochemical labelingwhich is demonstrated in Fig 1I and J Clearly the heparan sulfate proteoglycan ismore prevalent in the section derived from the pig than from the SIS This con rmsthe loss of some of the heparan sulfate proteoglycans during the processing

Growth peptides and low abundance components of SIS

Clearly the TSA procedure can identify and quantify components in tissues andSIS We next sought to apply it to components likely to be present in lowerconcentrations such as growth peptides which might not be evident against thebackground uorescence The distribution of FGF2 TGFmacr and VEGF in SIS wereinvestigated in relation to the amounts seen in pig intestine

Figure 4 illustrates a titration of the FGF2 antibody in the TSA system Thetwo points shown with the circle and square represent the controls which representan unrelated antibody of the same isotype as the primary used at the highestconcentration the speci c primary is used The lowest background values of thirdygray level values is still well below the speci c signal The values on the ordinate arearbitrary and are adjusted to keep the signal within the linear range of the measuring

Figure 4 Titration of anti-FGF2 antibody using TSA method for SIS (s) and pig intestine (u )(fF) represent an unrelated isotype-controlledmonoclonal antibody used as a negative control at thehighest concentrationused in the titration of the speci c antibody

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1276 R E Hurst and R B Bonner

Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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1272 R E Hurst and R B Bonner

Figure 1 Photographs of labeled SIS and pig intestine All photomicrographs are at the samemagni cation indicated by the bar in N (A) Double uorescent label of bronectin (green) andcollagen (red) in a 5-sup1m frozen SIS section demonstrating occasional preservation of morphology(B) A more usual result showing shredding and loss of morphology in a 5-sup1m frozen SIS sectionlabeled for heparan sulfate proteoglycan (green) and collagen (red) (C) Triple label of pig intestinedemonstrating speci city of labeling for bronectin (green) collagen (red) and DNA (blue) Thegreen area on the right is the villous epithelium The brown area is the submucosa from which SISis obtained (D) Labeling of collagen in a 20 sup1m section of SIS by indirect assay (E)ndash(H) Se-quential sections of pig intestine (E F) or SIS (G H) labeled for collagen by TSA ampli cation(E G) and heparan sulfate using a conventional sandwich method (F H) with 5 sup1m sections ofparaf n-preserved blocks Note the lack of background in areas where collagen or heparan sulfateis absent even with the heparan sulfate assay which does not employ the TSA ampli cation Theboundary between the submucosa and epithelium is at the bottom of the picture (E G) (I) and (J)Immunohistochemicallabelingof pig intestine(I) and SIS (J) for heparan sulfate proteoglycanin 5 sup1mparaf n block sections (K) and (L) FGF2 labelingby uorescence(K) and immunohistochemistry(L)demonstrating diffuse brous labeling Note FGF2 seems to be concentrated on the lumenal surface(top L) (M) and (N) VEGF labeling in pig intestine (M) and SIS (N) demonstrating localization tovessels and loss from SIS

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Figure 2 Titration of rabbit anticollagen (f) (positive) and (F)(negative control) in 20 sup1m frozensections of SIS using indirect immuno uorescence (sandwich) assay in comparison to TSA methodwith 5 sup1m sections The negative control substituted an unrelated serum at the same dilution as theprimary antibody The x-axis is arbitrary because the concentration of the polyclonal antibody wasunknown The difference between the positive and negative is proportional to the content of collagenin the sample The attening of the curve indicates saturation of binding sites and proportionality(s) shows the titration of the antibody using the TSA method on SIS and ( u ) on pig intestine Thedifferencebetween the two curves represents the difference in collagen immunoreactivitybetween thetwo specimens

wash buffers as well as the number of washes reduced the background uorescencebut never eliminated it In spite of these improvements consistent low backgroundcould not be obtained with the 20-sup1m sections Often speci c binding could beseen as bright areas against a low-intensity background These problems were ofleast signi cance for collagen which is highly abundant but were more signi cantfor markers that were of low abundance

Figure 2 illustrates the titration of the anti-collagen antibody with a conventionallsquosandwichrsquo assay on a 20-sup1m section of porcine intestine provided by Cook Biotechas being representative of the material from which SIS is made In the absenceof primary antibody there clearly is a background uorescence as shown bythe intercept on the y-axis The amount is the same with an unrelated primaryindicating that the background is due to failure to wash out all the uorophoreIncreasing the concentration of antibody yields the expected saturation of sites Inthe at portion of the curve the difference between background and the antibody-treated sample is directly proportional to the amount of target in this case collagen

Tyramide signal ampli cation (TSA) method

This technique offers a means to amplify the speci c signal arising from binding ofan individual antibody to its antigen in 5-sup1m paraf n sections thereby enhancingthe signal to noise ratio suf ciently to swamp out the nonspeci c uorescenceseen in paraf n sections This nonspeci c uorescence is not due to the antibody

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1274 R E Hurst and R B Bonner

because it appears in the absence of any reagents Instead of a uorochrome-labeledprimary or secondary the detection system consists of horseradish peroxidase(HRP) conjugated to the secondary antibody which then catalyzes deposition ofinsoluble biotin or dye-labeled tyramide in the immediate vicinity of the HRPAs each HRP moiety can generate numerous tyramide moieties a marked signalenhancement is observed The titration of antibody in the TSA system also isshown in Fig 2 The nonspeci c uorescence background is virtually undetectablein the controls for these assays of collagen which is a high-expression proteinand represents the ideal case for both conventional immuno uorescence assays andthe TSA ampli cation method The uorescence increases with primary antibodyconcentration from essentially zero indicating the ampli cation of the speci csignal over the nonspeci c signal As with the common lsquosandwichrsquo method thecurve breaks over indicating saturation of sites The difference in uorescenceintensity between the SIS and pig samples is proportional to the difference incollagen I content of the two samples

Heparan sulfate proteoglycans are of interest because they bind growth peptides invivo [18] The HS-PG is clearly detectable in the SIS material without ampli cation(Fig 1B F and H) Figure 3 shows the titration of heparan sulfate with theTSA method and demonstrates the presence of HS-PG in both the submucosalregion of the pig intestine and the SIS with the intestinal sample having a higherconcentration of the proteoglycan Figure 1EndashH shows the appearance of thecollagen enhanced by the TSA method and HS-PG labeled with a conventionallsquosandwichrsquo assay Collagen is labeled with red while heparan sulfate proteoglycanis labeled in green Figures 1E and F demonstrate the intact pig intestine whileFig 1G and H demonstrate SIS Note that the sections seem to be cut along differentplanes The glands and vessels are positive for heparan sulfate with very little

Figure 3 Titration of anti-heparan sulfate antibody using TSA method for SIS (s) and pigintestine (u )

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Molecular mapping of SIS 1275

background particularly in the SIS (Fig 1G and H) These high abundance proteinscould be quanti ed either from conventional immun uorescence even on paraf nsections or with the TSA method

With the TSA method negative controls are undetectable for measurement asare the lowest concentrations of this titration curve (Fig 3) Binding appears tobe very speci c and intense Both the unprocessed porcine intestine and the SISmaterial demonstrate speci c binding of anti-heparan sulfate antibody Vessels arethe primary positive component in the submucosa The values indicate that there issome loss of antigenicity (about 35) in SIS material compared to the intestinesample but clearly heparan sulfate is present within the body of the SIS itself(Fig 1H) The uorescence data are con rmed by immunohistochemical labelingwhich is demonstrated in Fig 1I and J Clearly the heparan sulfate proteoglycan ismore prevalent in the section derived from the pig than from the SIS This con rmsthe loss of some of the heparan sulfate proteoglycans during the processing

Growth peptides and low abundance components of SIS

Clearly the TSA procedure can identify and quantify components in tissues andSIS We next sought to apply it to components likely to be present in lowerconcentrations such as growth peptides which might not be evident against thebackground uorescence The distribution of FGF2 TGFmacr and VEGF in SIS wereinvestigated in relation to the amounts seen in pig intestine

Figure 4 illustrates a titration of the FGF2 antibody in the TSA system Thetwo points shown with the circle and square represent the controls which representan unrelated antibody of the same isotype as the primary used at the highestconcentration the speci c primary is used The lowest background values of thirdygray level values is still well below the speci c signal The values on the ordinate arearbitrary and are adjusted to keep the signal within the linear range of the measuring

Figure 4 Titration of anti-FGF2 antibody using TSA method for SIS (s) and pig intestine (u )(fF) represent an unrelated isotype-controlledmonoclonal antibody used as a negative control at thehighest concentrationused in the titration of the speci c antibody

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Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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Molecular mapping of SIS 1273

Figure 2 Titration of rabbit anticollagen (f) (positive) and (F)(negative control) in 20 sup1m frozensections of SIS using indirect immuno uorescence (sandwich) assay in comparison to TSA methodwith 5 sup1m sections The negative control substituted an unrelated serum at the same dilution as theprimary antibody The x-axis is arbitrary because the concentration of the polyclonal antibody wasunknown The difference between the positive and negative is proportional to the content of collagenin the sample The attening of the curve indicates saturation of binding sites and proportionality(s) shows the titration of the antibody using the TSA method on SIS and ( u ) on pig intestine Thedifferencebetween the two curves represents the difference in collagen immunoreactivitybetween thetwo specimens

wash buffers as well as the number of washes reduced the background uorescencebut never eliminated it In spite of these improvements consistent low backgroundcould not be obtained with the 20-sup1m sections Often speci c binding could beseen as bright areas against a low-intensity background These problems were ofleast signi cance for collagen which is highly abundant but were more signi cantfor markers that were of low abundance

Figure 2 illustrates the titration of the anti-collagen antibody with a conventionallsquosandwichrsquo assay on a 20-sup1m section of porcine intestine provided by Cook Biotechas being representative of the material from which SIS is made In the absenceof primary antibody there clearly is a background uorescence as shown bythe intercept on the y-axis The amount is the same with an unrelated primaryindicating that the background is due to failure to wash out all the uorophoreIncreasing the concentration of antibody yields the expected saturation of sites Inthe at portion of the curve the difference between background and the antibody-treated sample is directly proportional to the amount of target in this case collagen

Tyramide signal ampli cation (TSA) method

This technique offers a means to amplify the speci c signal arising from binding ofan individual antibody to its antigen in 5-sup1m paraf n sections thereby enhancingthe signal to noise ratio suf ciently to swamp out the nonspeci c uorescenceseen in paraf n sections This nonspeci c uorescence is not due to the antibody

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1274 R E Hurst and R B Bonner

because it appears in the absence of any reagents Instead of a uorochrome-labeledprimary or secondary the detection system consists of horseradish peroxidase(HRP) conjugated to the secondary antibody which then catalyzes deposition ofinsoluble biotin or dye-labeled tyramide in the immediate vicinity of the HRPAs each HRP moiety can generate numerous tyramide moieties a marked signalenhancement is observed The titration of antibody in the TSA system also isshown in Fig 2 The nonspeci c uorescence background is virtually undetectablein the controls for these assays of collagen which is a high-expression proteinand represents the ideal case for both conventional immuno uorescence assays andthe TSA ampli cation method The uorescence increases with primary antibodyconcentration from essentially zero indicating the ampli cation of the speci csignal over the nonspeci c signal As with the common lsquosandwichrsquo method thecurve breaks over indicating saturation of sites The difference in uorescenceintensity between the SIS and pig samples is proportional to the difference incollagen I content of the two samples

Heparan sulfate proteoglycans are of interest because they bind growth peptides invivo [18] The HS-PG is clearly detectable in the SIS material without ampli cation(Fig 1B F and H) Figure 3 shows the titration of heparan sulfate with theTSA method and demonstrates the presence of HS-PG in both the submucosalregion of the pig intestine and the SIS with the intestinal sample having a higherconcentration of the proteoglycan Figure 1EndashH shows the appearance of thecollagen enhanced by the TSA method and HS-PG labeled with a conventionallsquosandwichrsquo assay Collagen is labeled with red while heparan sulfate proteoglycanis labeled in green Figures 1E and F demonstrate the intact pig intestine whileFig 1G and H demonstrate SIS Note that the sections seem to be cut along differentplanes The glands and vessels are positive for heparan sulfate with very little

Figure 3 Titration of anti-heparan sulfate antibody using TSA method for SIS (s) and pigintestine (u )

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Molecular mapping of SIS 1275

background particularly in the SIS (Fig 1G and H) These high abundance proteinscould be quanti ed either from conventional immun uorescence even on paraf nsections or with the TSA method

With the TSA method negative controls are undetectable for measurement asare the lowest concentrations of this titration curve (Fig 3) Binding appears tobe very speci c and intense Both the unprocessed porcine intestine and the SISmaterial demonstrate speci c binding of anti-heparan sulfate antibody Vessels arethe primary positive component in the submucosa The values indicate that there issome loss of antigenicity (about 35) in SIS material compared to the intestinesample but clearly heparan sulfate is present within the body of the SIS itself(Fig 1H) The uorescence data are con rmed by immunohistochemical labelingwhich is demonstrated in Fig 1I and J Clearly the heparan sulfate proteoglycan ismore prevalent in the section derived from the pig than from the SIS This con rmsthe loss of some of the heparan sulfate proteoglycans during the processing

Growth peptides and low abundance components of SIS

Clearly the TSA procedure can identify and quantify components in tissues andSIS We next sought to apply it to components likely to be present in lowerconcentrations such as growth peptides which might not be evident against thebackground uorescence The distribution of FGF2 TGFmacr and VEGF in SIS wereinvestigated in relation to the amounts seen in pig intestine

Figure 4 illustrates a titration of the FGF2 antibody in the TSA system Thetwo points shown with the circle and square represent the controls which representan unrelated antibody of the same isotype as the primary used at the highestconcentration the speci c primary is used The lowest background values of thirdygray level values is still well below the speci c signal The values on the ordinate arearbitrary and are adjusted to keep the signal within the linear range of the measuring

Figure 4 Titration of anti-FGF2 antibody using TSA method for SIS (s) and pig intestine (u )(fF) represent an unrelated isotype-controlledmonoclonal antibody used as a negative control at thehighest concentrationused in the titration of the speci c antibody

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1276 R E Hurst and R B Bonner

Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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1274 R E Hurst and R B Bonner

because it appears in the absence of any reagents Instead of a uorochrome-labeledprimary or secondary the detection system consists of horseradish peroxidase(HRP) conjugated to the secondary antibody which then catalyzes deposition ofinsoluble biotin or dye-labeled tyramide in the immediate vicinity of the HRPAs each HRP moiety can generate numerous tyramide moieties a marked signalenhancement is observed The titration of antibody in the TSA system also isshown in Fig 2 The nonspeci c uorescence background is virtually undetectablein the controls for these assays of collagen which is a high-expression proteinand represents the ideal case for both conventional immuno uorescence assays andthe TSA ampli cation method The uorescence increases with primary antibodyconcentration from essentially zero indicating the ampli cation of the speci csignal over the nonspeci c signal As with the common lsquosandwichrsquo method thecurve breaks over indicating saturation of sites The difference in uorescenceintensity between the SIS and pig samples is proportional to the difference incollagen I content of the two samples

Heparan sulfate proteoglycans are of interest because they bind growth peptides invivo [18] The HS-PG is clearly detectable in the SIS material without ampli cation(Fig 1B F and H) Figure 3 shows the titration of heparan sulfate with theTSA method and demonstrates the presence of HS-PG in both the submucosalregion of the pig intestine and the SIS with the intestinal sample having a higherconcentration of the proteoglycan Figure 1EndashH shows the appearance of thecollagen enhanced by the TSA method and HS-PG labeled with a conventionallsquosandwichrsquo assay Collagen is labeled with red while heparan sulfate proteoglycanis labeled in green Figures 1E and F demonstrate the intact pig intestine whileFig 1G and H demonstrate SIS Note that the sections seem to be cut along differentplanes The glands and vessels are positive for heparan sulfate with very little

Figure 3 Titration of anti-heparan sulfate antibody using TSA method for SIS (s) and pigintestine (u )

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Molecular mapping of SIS 1275

background particularly in the SIS (Fig 1G and H) These high abundance proteinscould be quanti ed either from conventional immun uorescence even on paraf nsections or with the TSA method

With the TSA method negative controls are undetectable for measurement asare the lowest concentrations of this titration curve (Fig 3) Binding appears tobe very speci c and intense Both the unprocessed porcine intestine and the SISmaterial demonstrate speci c binding of anti-heparan sulfate antibody Vessels arethe primary positive component in the submucosa The values indicate that there issome loss of antigenicity (about 35) in SIS material compared to the intestinesample but clearly heparan sulfate is present within the body of the SIS itself(Fig 1H) The uorescence data are con rmed by immunohistochemical labelingwhich is demonstrated in Fig 1I and J Clearly the heparan sulfate proteoglycan ismore prevalent in the section derived from the pig than from the SIS This con rmsthe loss of some of the heparan sulfate proteoglycans during the processing

Growth peptides and low abundance components of SIS

Clearly the TSA procedure can identify and quantify components in tissues andSIS We next sought to apply it to components likely to be present in lowerconcentrations such as growth peptides which might not be evident against thebackground uorescence The distribution of FGF2 TGFmacr and VEGF in SIS wereinvestigated in relation to the amounts seen in pig intestine

Figure 4 illustrates a titration of the FGF2 antibody in the TSA system Thetwo points shown with the circle and square represent the controls which representan unrelated antibody of the same isotype as the primary used at the highestconcentration the speci c primary is used The lowest background values of thirdygray level values is still well below the speci c signal The values on the ordinate arearbitrary and are adjusted to keep the signal within the linear range of the measuring

Figure 4 Titration of anti-FGF2 antibody using TSA method for SIS (s) and pig intestine (u )(fF) represent an unrelated isotype-controlledmonoclonal antibody used as a negative control at thehighest concentrationused in the titration of the speci c antibody

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1276 R E Hurst and R B Bonner

Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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Molecular mapping of SIS 1275

background particularly in the SIS (Fig 1G and H) These high abundance proteinscould be quanti ed either from conventional immun uorescence even on paraf nsections or with the TSA method

With the TSA method negative controls are undetectable for measurement asare the lowest concentrations of this titration curve (Fig 3) Binding appears tobe very speci c and intense Both the unprocessed porcine intestine and the SISmaterial demonstrate speci c binding of anti-heparan sulfate antibody Vessels arethe primary positive component in the submucosa The values indicate that there issome loss of antigenicity (about 35) in SIS material compared to the intestinesample but clearly heparan sulfate is present within the body of the SIS itself(Fig 1H) The uorescence data are con rmed by immunohistochemical labelingwhich is demonstrated in Fig 1I and J Clearly the heparan sulfate proteoglycan ismore prevalent in the section derived from the pig than from the SIS This con rmsthe loss of some of the heparan sulfate proteoglycans during the processing

Growth peptides and low abundance components of SIS

Clearly the TSA procedure can identify and quantify components in tissues andSIS We next sought to apply it to components likely to be present in lowerconcentrations such as growth peptides which might not be evident against thebackground uorescence The distribution of FGF2 TGFmacr and VEGF in SIS wereinvestigated in relation to the amounts seen in pig intestine

Figure 4 illustrates a titration of the FGF2 antibody in the TSA system Thetwo points shown with the circle and square represent the controls which representan unrelated antibody of the same isotype as the primary used at the highestconcentration the speci c primary is used The lowest background values of thirdygray level values is still well below the speci c signal The values on the ordinate arearbitrary and are adjusted to keep the signal within the linear range of the measuring

Figure 4 Titration of anti-FGF2 antibody using TSA method for SIS (s) and pig intestine (u )(fF) represent an unrelated isotype-controlledmonoclonal antibody used as a negative control at thehighest concentrationused in the titration of the speci c antibody

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1276 R E Hurst and R B Bonner

Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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Figure 5 Analysis of different batches of SIS for FGF2 Samples 2 and 4 were analyzed in triplicateand are indicated by dotted patterns The error bars represent the standard deviations observed withmeasurements of ten different elds The two bars on the right show results obtainedwith an unrelatedprimary antibody of the same isotype as the speci c antibody

system Because FGF2 and other low-abundance proteins are less abundant thancollagen or the heparan sulfates the background appears to be more signi cant thanis seen with the more abundant proteins In this example which is one of severalexperiments performed the SIS sections analyzed were more immunoreactive withanti-FGF2 than was the pig section However as is shown in Fig 1K and L theFGF2 immunoreactivity is diffusely and heterogeneously distributed and outlines brous material Thus the amount measured will depend upon where the sectionsare obtained In other examples the opposite is observed as is shown in Fig 5which shows analyses of a series of different samples of SIS In these data mostof the SIS samples contain less FGF2 than the pig sample but some contain moreThree replicates determined from different though adjacent sections of the samesamples also are shown and indicated by a different pattern The two samples onthe far right represent labeling with an unrelated antibody of the same isotype as theprimary used to label FGF2

TGFmacr is a major effector of stromalndashepithelial interactions It normally inhibitsgrowth of epithelial cells but stimulates growth of stromal cells While the signalwas small generally there was more in the intestine than in the SIS indicatinga loss of TGFmacr from the particular preparation of SIS analyzed Interestinglythe background uorescence was consistently higher with the intestine Howeverthese measurements represent the absolute limit of the assay as it is currentlycon gured The speci city of the signal over background is illustrated in Fig 6The immunoreactivity is diffusely distributed but because the photographic process

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Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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Molecular mapping of SIS 1277

Figure 6 Titration of anti-TGFmacr1 antibody using TSA method (s) D SIS (u ) D pig intestine

does not capture the quantitative difference from the negative controls a photographis not shown

VEGF represents another highly angiogenic peptide that might affect the growthof cells and structures within the regenerating bladder Figure 1M and N demon-strates that the VEGF seems to be reduced during the processing The VEGF thatwas present in the original pig intestine surrounding the vessels is reduced in theSIS but examination of Fig 1N demonstrates the signal still is present Because thevessels seem to have the highest levels of auto uorescence it was not possible toobtain accurate measurements of VEGF by uorescence

DISCUSSION

In this study the distribution of matrix proteins and growth peptides was inves-tigated in the bioscaffold material SIS A new sensitive uorescence method ap-plicable to paraf n sections was adapted to provide relative quantitation of even lowabundance biomolecules such as growth peptides Examination of titration curvesallows the relative amounts of these proteins to be compared in different samplematerials measured at the same time These studies demonstrate the principle ofanalysis using QFIA rather than providing a comprehensive analysis of differentbatches of SIS although analyses of a series of samples for FGF2 is presented Thedata show that SIS contains growth peptides some of which are reduced duringprocessing and that some of these are distributed along certain anatomic structuresThe replication inherent in these curves provides considerable con dence that thedifferences are real and reproducible

The assays using immuno uorescence do suffer from certain limitations mostnotably that the epitope must be available to the antibody In order to minimize suchproblems most assays were performed using at least two different antibodies Weattempted where possible to use at least one polyclonal immunoadsorbed antiseraor immunoglobin preparation The variety of epitopes seen by antiserum-derivedantibodies minimizes the potential masking of epitopes that might occur with amonoclonal that binds to a single speci c epitope Also assays were comparedwith and without antigen retrieval Generally there was little difference between the

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results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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1278 R E Hurst and R B Bonner

results with and without such treatment That TGFmacr was barely detected under theseconditions in spite of having been identi ed in biochemical analyses followinglimited proteolysis [10] suggests that it is very diffusely distributed with a lowlocal concentration

The most signi cant ndings were that some of the factors are reduced in SIS ascompared to the original pig intestine and that some seem to be concentrated inthe remnant vessels The loss of components suggests that different batches of SISmay well vary in the content of these components Additionally there may wellbe biological variation in the distribution of these peptide components along thelength of the intestine The anatomic structure of SIS is likely to play an importantrole in the regeneration of tissues The remnant vessels contain a number of factorsthat might facilitate penetration of the SIS matrix along these avenues In factobservations of the growth of cells on SIS shows that the growing cells penetrate andgrow preferentially along remnant vessels [9 19] However the possibility must beconsidered that the connection of invading smooth muscle and urothelial cells tothe matrix itself is the key to its bioscaffold activity Thus factors in the remnantvessels may facilitate the rapid spread of cells which then form connections to thematrix which then guides subsequent growth and differentiation along with cell-cellinteractions and new matrix formation

CONCLUSIONS

The methods we present here should be useful for monitoring events involvedafter implantation in animals or humans by obtaining biopsies during the courseof regeneration as well as for investigating the composition of SIS The ndings ofsigni cant levels of growth peptides and proteins other than collagen suggest thesemay play a role in its bioscaffold activity

Acknowledgements

This work was supported by a grant from Cook Biotech Inc and from the NationalCancer Institute CA 75322 The authors thank Jean Coffman for excellent technicalassistance

REFERENCES

1 B P Kropp J K Ludlow D Spicer M K Rippy S F Badylak M C Adams M A KeatingR C Rink R Birhle and K B Thor Urology 52 138 (1998)

2 M A Cobb S F Badylak W Janas and F A Boop Surg Neurol 46 389 (1996)3 B P Kropp M K Rippy S F Badylak M C Adams M A Keating R C Rink and K B Thor

J Urol 155 2098 (1996)4 B P Kropp B D Sawyer H E Shannon M K Rippy S F Badylak M C Adams

M A Keating R C Rink and K B Thor J Urol 156 599 (1996)

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5 M C Hiles S F Badylak G C Lantz K Kokini L A Geddes and R J Morff J BiomedMater Res 29 883 (1995)

6 B P Kropp B L Eppley C D Prevel M K Rippy R C Harruff S F Badylak M C AdamsR C Rink and M A Keating Urology 46 396 (1995)

7 B P Kropp S Badylak and K B Thor Adv Exp Med Biol 385 229 (1995)8 G E Sandusky G C Lantz and S F Badylak J Surg Res 58 415 (1995)9 S F Badylak R Record K Lindberg J Hodde and K Park J Biomater Sci Polymer Edn 9

863 (1998)10 S L Voytik-Harbin A O Brightman M R Kraine B Waisner and S F Badylak J Cell

Biochem 67 478 (1997)11 R B Bonner R E Hurst J Rao and G P Hemstreet in Tumor Marker Protocols M Hanausek

and Z Walaszek (Eds) p 181 Humana Press Totowa NJ (1998)12 G P Hemstreet R E Hurst and R B Bonner in Tumor Marker Protocols M Hanausek and

Z Walaszek (Eds) p 37 Humana Press Totowa NJ (1998)13 R B Bonner M Liebert R E Hurst H B Grossman B L Bane and G P Hemstreet Cancer

Epidemiol Biomarkers Prev 5 971 (1996)14 R B Bonner G P Hemstreet Y Fradet J Y Rao K W Min and R E Hurst Cancer 72 2461

(1993)15 J Y Rao G P Hemstreet R E Hurst R B Bonner P L Jones K W Min and Y Fradet Proc

Natl Acad Sci USA 90 8287 (1993)16 P McGowan R E Hurst R E Bass G P Hemstreet and R Postier J Histochem Cytochem

36 757 (1988)17 J Y Rao R B Bonner R E Hurst W R Qiu C A Reznikoff and G P Hemstreet Int

J Cancer 70 423 (1997)18 I Vlodavsky H Q Miao B Medalion P Danagher and D Ron Cancer Metastasis Rev 15

177 (1996)19 S L Voytik-Harbin A O Brightman B Z Waisner J P Robinson and C H Lamar Tissue

Eng 4 157 (1998)

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