TISSUE ENGINEERINGVolume 4, Number 1, 1998Mary Ann Liebert, Inc.

Characterization of Fibronectin Derived from PorcineSmall Intestinal Submucosa

TIMOTHY B. McPHERSON, Ph.D. and STEPHEN F. BADYLAK, D.V.M., Ph.D., M.D.

ABSTRACT

Small intestinal submucosa (SIS) is an acellular biomaterial derived from porcine jejunum.When used as a soft tissue graft material, SIS induces site-specific remodeling of the organor tissue in which it is placed. The mechanism by which SIS induces tissue remodeling isonly partially understood. Fibronectin (Fn) is a dimeric glycoprotein present in plasma andextracellular matrix. Fn exhibits chemotactic and adhesive properties for many cells, in-cluding fibroblasts and endothelial cells. Thus, Fn may play a key role in the tissue-remod-eling activity of SIS. The goals of this study were to localize and quantify the Fn in SIS, andcharacterize the structure of SIS-derived Fn. Immunohistochemical staining confirmed thetransmural presence of Fn in SIS. The Fn content of SIS was 0.08 ± 0.05% dry weight, sim-ilar to the Fn content of other similar tissues, as determined with a competitive ELISA. SDS-PAGE and Western blots of purified protein showed SIS-derived Fn to migrate similar tohuman and porcine plasma fibronectins. Fn derived from SIS did not contain domains as-sociated with embryonic or transitional extracellular matrix. Fn is transmurally distributedthroughout the thickness of SIS and its content and structure are consistent with stable, ma-ture tissue.

INTRODUCTION

SMALL INTESTINAL SUBMUCOSA (SIS) is a naturally occurring biomaterial derived from the jejunum ofmammals, most commonly pigs. SIS is acellular after minimal processing and consists solely of the ex-

tracellular matrix. When used as a xenogeneic graft material, SIS induces specific tissue remodeling in theorgan or tissue into which it is placed. It has been successfully used in vascular grafts; hernia and urinarybladder repair; and orthopedic, dermal graft, and dura mater applications.1"8 The remodeled tissue resem-bles native tissue both grossly and microscopically. All evidence suggests that the originally implanted SISis resorbed and replaced by host tissue within 90 days. The mechanism by which SIS induces tissue re-modeling is only partially understood.

Fibronectin (Fn) is a dimeric glycoprotein found in the extracellular matrix and plasma and has a mole-cular mass of approximately 440 kDa. Fn exhibits binding affinities for various molecules including gelatin.

Hillenbrand Biochemical Engineering Center, Purdue University, West Lafayette, Indiana 47907.A poster describing some of this work was presented at the Tissue Engineering Meeting in Orlando, Florida in De-

cember 1996.

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McPHERSON AND BADYLAK

fibrinogen, heparin, thrombospondin, complement Clq, plasminogen, and tissue plasminogen activator.910

The structure of Fn consists of three types (I, II, III) of repeated subunits connected by flexible regions. Fnvariants, which appear to be cell type and environment specific, are spliced from a single gene. Two vari-ably expressed extra type III repeats associated with cellular forms of Fn, referred to as EIIIA and EIIIB,may be included in the molecule. A variable segment (V) with a structure unrelated to the three main typesmay be included in whole or in part. Cellular Fn typically contains V regions, whereas about 50% of plasmaFn contains V regions. The EIIA and EIIB domains are prevalent during embryonic development and woundhealing, but are generally absent from plasma and stable, mature tissue.11

Fn is among the first proteins deposited in new extracellular matrix in wound healing,12 and exhibits ad-hesive and chemotactic properties for a variety of cell types, including endothelial cells and fibroblasts. Asthese cell types are important to the wound-healing process,12 Fn may play a pivotal role in the observedin vivo remodeling response. Preliminary work revealed that Fn is present in SIS (our unpublished data,1995). The aims of the present study were to localize Fn within the SIS, quantify the amount of Fn presentin the tissue, and characterize the structure of SIS-derived Fn.

MATERIALS AND METHODS

Small Intestinal Submucosa Preparation

SIS was prepared as described elsewhere by Badylak and others.1 Briefly, fresh porcine jejunum, ob-tained from a local slaughterhouse, was inverted and the superficial layers of the tunica mucosa removed.The tissue was then reverted to its original orientation and the serosa and tunica muscularis removed. Theresulting membrane, which is 80-100 /xm thick, consists of the tunica submucosa and the basilar portionof the tunica mucosa.

Scanning Electron Microscopy

Samples of SIS were fixed in 10% neutral buffered formalin, dehydrated in a graded ethanol series, crit-ical point dried with carbon dioxide, and mounted on an aluminum stub. Samples were sputtered with agold-palladium target and observed on a scanning electron microscope (JSM-840; JEOL, Tokyo, Japan) setto an accelerating voltage of 5 kV.

Immunohistochemical Localization of Fibronectin

SIS and whole porcine intestine were frozen in O.C.T. (Miles, Elkhart, IL) embedding medium, sectionedon a cryomicrotome set to a cutting thickness of 7 /xm, mounted on poly-L-lysine-coated slides, and airdried. The tissues were fixed in acetone at 4°C for 3 min and blocked in 5% goat serum (Sigma ChemicalCompany, St. Louis, MO) in phosphate-buffered saline (PBS), pH 7.4, for 20 min. Endogenous peroxidaseactivity was inhibited by incubation for 30 min in H2O2 (0.45%, v/v) in methanol. Rabbit anti-chicken Fnserum (Chemicon International, Temecula, CA) or control rabbit IgG (purified from normal rabbit serum)was applied to the tissue for 1 h, followed by peroxidase-conjugated goat anti-rabbit IgG (Sigma) for 1 h.The slides were then exposed to peroxidase substrate solution(3,3'-diaminobenzidine [0.06%] and H2C»2[0.015%, v/v] in 50 mM Tris [pH 7.6]) for 10 min. All incubations were done at room temperature unlessotherwise specified, and slides were rinsed in PBS for 10 min between incubations. The tissues were coun-terstained with hematoxylin, dehydrated in a graded ethanol series, and mounted with a coverslip.

Extraction of Fibronectin from Small Intestinal Submucosa

Frozen SIS was ground in an industrial blender (Waring Products, New Hartford, CT) under liquid ni-trogen and weighed into a tared flask. Extraction buffer (2 M urea, heparin [2.5 mg/ml], 50 mM Tris [pH7.5]) was added to the flask at 4 ml/g of tissue. Phenylmethylsulfonyl fluoride (Sigma), /V-ethylmaleimide(Sigma), and benzamidine (Sigma) were each added to a final concentration of 1 mM. The mixture wasstirred for 18 h at 4°C. The remaining tissue was pelleted at 12,000 X g for 30 min at 4°C. The supernatantwas stored at 4°C and the tissue pellet was extracted in fresh buffer for 18 h. After the second extraction,

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CHARACTERIZATION OF FIBRONECTIN

the tissue was centrifuged at 27,200 X g for 30 min at 4°C to form a pellet. The supernatant was collectedand the tissue pellet extracted for four more hours. After collecting the third supernatant, the remaining tis-sue was digested with collagenase (type VII from Closthdium histolyticum [Sigma]) 50 units/ml in Tris-buffered saline 1 mM CaCl2 pH 7.4 at room temperature for 18 h. This final suspension was centrifugedand the supernatant retained. Supernatants were dialyzed against Tris buffered saline, pH 7.4.

Fibronectin Quantification

A competitive enzyme-linked immunosorbent assay (ELISA) based on published protocols13"15 was de-veloped to quantify Fn in extracts of SIS. All incubations were done at room temperature, except as noted,and the sample wells were appropriately rinsed between incubations with 0.05% Tween 20 in Tris-bufferedsaline (TBS), pH 7.4. Sample wells of high protein-binding microtiter plates were coated with 100 fi\ ofporcine plasma Fn (purified as described below), 50 Aig/ml, at 4°C overnight. Twofold serial dilutions ofstandard porcine plasma Fn (50 /xg/ml) and SIS extracts were prepared in wells of a separate plate that werepreviously blocked with 5% bovine serum albumin for 1 h. Rabbit anti-chicken Fn (primary Ab) was addedto each sample to a final dilution of 1:1000. Samples were prepared in triplicate and incubated at 4°Covernight.

Controls consisted of wells containing primary Ab alone and standard Fn alone. The wells of the testplate were rinsed of the Fn coating solution and blocked with bovine serum albumin (BSA) at 5 mg/ml.The sample mixtures were transferred to the Fn-coated plate at 100 /i.l/well and incubated for 2 h. Duringthis incubation, Fn in solution competes with surface-bound Fn for binding of the primary Ab. Goat anti-rabbit IgG peroxidase conjugate (secondary Ab) was then added to label the surface-bound primary Ab.After 1 h incubation, the chromogenic peroxidase substrate solution (ABTS; Kirkegaard & Perry Labora-tories, Gaithersburg, MD) was added. The reaction was stopped after 40 min by the addition of 100 /xl of\% sodium dodecyl sulfate (SDS; Sigma). The color intensity was read in a plate reader (Bio-Tek Instru-ments, Winooski, VT) at 405 nm. The data were converted to percent inhibition of primary Ab binding ver-sus log soluble Fn concentration, using the linear portion of the standard curves.

SDS-PAGE and Western Blot Analysis

SIS-derived tissue Fn (tFn) was purified by gelatin affinity chromatography.16 Porcine plasma Fn (ppFn)and human plasma Fn (hpFn) were similarly purified from citrated blood. SDS- polyacrylamide gel elec-trophoresis (SDS-PAGE) of tFN, ppFn, and hpFn was conducted under reducing conditions on a 4-15%gradient gel (Bio-Rad, Hercules, CA), and stained with Coomassie Brilliant Blue R250 (Bio-Rad). Proteinwas loaded at 4 /itg/lane for stained gels, and 0.25 yug/lane for blots. Fn samples to be blotted with anti-EIIB Ab were digested with PNGase F (New England BioLabs, Beverly, MS) as described17 before elec-trophoresis.

For Western blot analyses, proteins were transferred to nitrocellulose at 10 V for 30 min, blocked in 5%dry milk, and detected with polyclonal rabbit anti-Fn serum; monoclonal mouse anti-cellular Fn (Sigma),which recognizes the EIIA domain; or affinity-purified rabbit anti-Fn IgG, which recognizes the EIIB do-main (generously provided by R. Hynes, Massachusetts Institute of Technology). Goat anti-rabbit IgG oranti-mouse IgM peroxidase conjugates were then applied, followed by enhanced chemiluminescence (ECL;Amersham, Arlington Heights, IL) substrate. Blots were rinsed between incubations with 0.05% Tween 20in TBS.

RESULTS

SIS Structure

The structure of SIS is shown in Figure 1. The lumenal surface is a smooth layer, made up of denselypacked, highly organized fibers (Figure 1 A). At least some of the pores that penetrate this layer are thoughtto represent remnants of capillaries or lymphatic vessels, the lining of which have been removed during thepreparation of SIS. The ablumenal surface (Figure IB) is much less dense than the lumenal surface, con-

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McPHERSON AND BADYLAK

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FIG. 1. Scanning electron micrograph of lumenal (A) and ablumenal (B) surfaces of SIS. Bar: 100 /jum.

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CHARACTERIZATION OF FIBRONECTIN

B

FIG. 2. SIS immunoperoxidase stained with rabbit anti-Fn serum (A) showing homogeneous transmuralstaining with greater intensity at lumenal surface (arrow), and SIS identically treated with control rabbitIgG (B) showing no staining. Original magnification: X74.

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McPHERSON AND BADYLAK

sisting of several layers of fibrous matrix. This layer is also highly organized, however, with the fibers ori-ented in the same direction as those of the lumenal surface.

Immunohistochemistry

Immunohistochemical staining showed that Fn was present in a transmural distribution throughout thethickness of SIS (Figure 2A). The greater intensity at the lumenal surface (arrow) may be due solely to thegreater tissue density of this layer (Figure 1). SIS treated with the control rabbit Ab showed no substrateconversion (Figure 2B), confirming the specificity of Fn staining. The faint background color in Figure 2Bis due to hematoxylin counterstain. Whole pig intestine showed a transmural specific Fn staining pattern inthe area from which SIS is derived, and no staining when treated with the control Ab (not shown).

ELISA

The dry weight of SIS after lyophilization was 8.2% of the starting weight. Thus, a total of 725 mg ofdry SIS was extracted for Fn analysis. Quantitative ELISA revealed that a significant amount of Fn was ex-tracted from SIS with urea and heparin. Representative ELISA curves are shown in Figure 3. The first ex-tract contained the greatest amount of Fn, at 8.8 /Jig/ml. The Fn concentration decreased in the second andthird extractions, as expected, to 1.0 and 0.2 /xg/ml, respectively. Finally, the collagenase digest containedFn at 0.6 /xg/ml. The data were multiplied by the respective extract volumes to obtain the total Fn yieldfrom the SIS. Assuming that all Fn was solubilized by the serial extraction18 and enzyme digestion, Fn rep-resents 0.08 ± 0.05% of the dry weight of SIS.

SDS-PAGEIWestern Blot

SDS-PAGE (Figure 4) showed that Fn purified from porcine SIS migrates similarly to both human andporcine plasma forms, as the expected mass of slightly greater than 200 kDa. The tFn band was resolvedinto a doublet, which indicates at least two Fn chain variants are present. Western blotting with rabbit poly-clonal anti-Fn serum detected all three Fn forms (Figure 5). The low molecular weight bands in the ppFnand tFn lanes suggest greater variability in the Fn or the presence of proteolytic fragments. The mouse anti-

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FIG. 3. Representative ELISA percent inhibition versus reciprocal dilution of Fn standard and SIS ex-tracts. Mean ± standard deviation of triplicate wells. Fn standard, 50 jug/ml ( • ) ; extract 1 ( • ) ; extract 2( • ) ; extract 3 (A); collagenase digest (T).

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CHARACTERIZATION OF FIBRONECTIN

FIG. 4. Coomassie Blue-stained SDS-polyacrylamide gel of purified fibronectins. Lane 1, molecularweight standards; lane 2, tFn; lane 3, ppFn; lane 4, hpFn.

EIIIA and rabbit anti-EIIIB antibodies did not detect any of the Fn samples (not shown). Thus, it appearsthat SIS-derived tFn contains neither of the cellular extra domains.

DISCUSSION

SIS consists of a mixture of structural and functional proteins arranged in an ordered and organized archi-tecture. The acellular, fibrous network is an excellent substrate for cell growth in vivo. The known compo-nents of SIS include collagen,6 glycosaminoglycans,19 the growth factors TGF-/3 and basic FGF,20 and theglycoproteins laminin and entactin (our unpublished data, 1995). The effect of the biochemical componentsand structural properties on the wound-healing activities of SIS is only partially understood at the present time.

FIG. 5. Western blot of purified fibronectins detected with anti-Fn serum. Lane 1, tFn; lane 2, ppFn; lane3, hpFn.

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TABLE 1. REPORTED VALUES FOR FIBRONECTIN CONTENT OF TISSUES

Tissue Fn content (% dry wt) Ref.

Placental villi 1.8-2.9 13Chicken tendon

Epitenon 0.6 ± 0.009 21Sheath synovium 0.118 ± 0.022Tendon 0.016 ± 0.001

Mouse glomerular basement membrane 0.82 ± 0.08 22Rat renal cortex 0.69 ± 0.1 23

This study confirms the presence of Fn in SIS. That Fn is transmurally distributed in SIS is not surpris-ing, considering the role of Fn in extracellular matrix. Literature values for Fn content of various tissuesare listed in Table l.13-21"23 The Fn content of SIS, 0.08 ± 0.05%, is in agreement with these values. Therelatively large standard deviation is the result of determining Fn concentration from a log-linear standardcurve, which exponentially magnifies minor deviations when converting to concentration values.

The structure of SIS-derived Fn was not demonstrated to contain EIIIA or EIIIB domains. The lack ofthese domains in SIS may be useful in studying the in vivo remodeling response. SIS appears to be gradu-ally resorbed after implantation, on the basis of immunohistochemical staining with SIS-specific mono-clonal antibody.6-24 Thus, new Fn containing EIIIA or EIIIB domains in the remodeling tissue would sug-gest that it was deposited by the host cells rather than originating in SIS. Variable domains were not assayedfor in this study. Because it is reported that most cellular Fn contains these domains, they would not likelybe useful in discriminating host tissue from SIS during remodeling.

Cell binding to Fn is mediated by integrins, heterodimeric integral membrane proteins that bridge the ex-tracellular matrix and cytoskeleton. Integrins also mediate the interaction of cells with other adhesive ex-tracellular matrix proteins such as laminin and vitronectin.9 Laminin has been demonstrated in SIS (our un-published data, 1995), and the presence of vitronectin is currently being studied. The integrin-mediatedchemotactic and adhesive activities of Fn, in concert with other proteins, may contribute to the favorabletissue response to SIS by inducing host cells to migrate into the acellular graft. The early angiogenic re-sponse to SIS in vivo1'2 suggests such chemotactic recruitment of microvascular endothelial cells. Fn likelyacts to anchor cells to the matrix and induce spreading, facilitating their interaction with the graft and di-recting new matrix synthesis.12 The relative contributions of Fn, laminin, and other proteins, and the pos-sible interactions between them, are not known. In addition to its direct effects on cells, Fn binds trans-forming growth factor /3.12 Thus, Fn may promote remodeling of SIS by recruiting and retaining host cellsin the graft, and by modulating growth factor activity.

ACKNOWLEDGMENTS

We gratefully acknowledge Mr. Jason Hodde for the initial identification of glycoproteins in SIS, Dr.Steve Nail (Department of Industrial and Physical Pharmacy, Purdue University School of Pharmacy) forthe use of the microplate reader and chromatography system, and Dr. Richard Hynes (Massachusetts Insti-tute of Technology) for providing the anti-EIIIB antibody. This work was supported by Cook Biotech, Inc.,the Trask Fund of Purdue University, and the National Institutes of Health (HD 31425).

REFERENCES

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2. Sandusky, G.E., Badylak, S.F., Morff, R.J., Johnson, W.D., and Lantz, G. Histologic findings after in vivo place-ment of small intestine submucosal vascular grafts and saphenous vein grafts in the carotid artery in dogs. Am. J.Pathol. 140, 317, 1992.

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3. Clarke, K.M., Lantz, G.C, Salisbury, S.K., Badylak, S.F., Hiles, M.C., and Voytik, S.L. Intestine submucosa andpropylene mesh for abdominal wall repair in dogs. J. Surg. Res. 60, 107, 996.

4. Knapp, P.M., Lingeman, J.E., Siegel, Y.I., Badylak, S.F., and Demeter, R.J. Biocompatibility of small intestinalsubmucosa in urinary tract as augmentation cytoplasty graft and injectable suspension. J. Endourol. 8, 125, 1994.

5. Kropp, B.P., Eppley, B.L., Prevel, CD., Rippy., M.K. Harruff, R.C., Badylak, S.F., Adams, M.D., Rink, R.C., andKeating, M. A. Experimental assessment of small intestine submucosa as a bladder wall substitute. Urology 46, 396,1995.

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bronectin contain binding domains for human plasminogen and tissue plasminogen activator. J. Biol. Chem.268, 18917. 1992.

11. Yamada, K.M., and Clark, R.A.F. Provisional matrix. In Clark, R., ed. The Molecular and Cellular Biologyof Wound Repair. New York: Plenum Press, 1996, pp. 51-93.

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plasma fibronectin. J. Immunol. Methods 40, 101, 1981.15. Rennard, S.I., Berg, R., Martin, G.R., Foidart, F.M., and Robey, P.G. Enzyme-linked immunoassay (ELISA)

for connective tissue components. Anal. Biochem. 104, 205, 1980.16. Vuento, M., and Vaheri, A. Purification of fibronectin from human plasma by affinity chromatography un-

der non-denaturing conditions. Biochem. J. 183, 331, 1979.17. Peters, J.H., Trevithick, J.E., Johnson, P., and Hynes, R.O. Expression of the alternatively spliced EIIIB seg-

ment of fibronectin. Cell Adh. Commun. 3, 1995.18. Bray, B.A., Mandl, U., and Turino, G.M. Heparin facilitates the extraction of tissue fibronectin. Science 214,

793, 1981.19. Hodde, J.P., Badylak, S.F., Brightman, A.O., and Voytik-Harbin, S.L. Glycosaminoglycan content of small

intestinal submucosa: A bioscaffold for tissue replacement. Tissue Eng. 2, 209, 1996.20. Voytik-Harbin, S.L., Brightman, A.O., Kraine, M., Waisner, B., and Badylak, S.F. Identification of FGF-2

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21. Brigman, B.E., Hu, P., Yin, H., Tsuzaki, M., Lawrence, W.T., and Banes, A.J. Fibronectin in the tendon-syn-ovial complex: Quantitation in vivo and in vitro by ELISA and relative mRNA levels by polymerase chainreaction and Northern blot. J. Orthoped. Res. 12, 253, 1994.

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24. Hiles, M.D., Badylak, S.F., Lantz, G.C., Kokini, K., Geddes, L.A., and Morff, R.J. Mechanical properties ofxenogenetic small-intestinal submucosa when used as an aortic graft in the dog. J. Biomed. Mater. Res. 29,883, 1995.

Address reprint requests to:Timothy B. McPherson

Hillenbrand Biomedical Engineering Center1293 Potter Bldg.Purdue University

West Lafayette, Indiana 47907

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