Layer-by-Layer Incorporation of Growth Factors inDecellularized Aortic Heart Valve Leaflets

Liesbeth J. De Cock,† Stefaan De Koker,‡ Filip De Vos,§ Chris Vervaet,†

Jean-Paul Remon,† and Bruno G. De Geest*,†

Laboratory of Pharmaceutical Technology, Department of Pharmaceutics, Ghent University,Harelbekestraat 72, 9000 Ghent, Belgium, Department of Molecular Biomedical Research, Ghent

University, Technologiepark Zwijnaarde 927, 9052 Ghent, Belgium, and Laboratory of Radiopharmacy,Department of Pharmaceutical Analysis, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium

Received December 23, 2009; Revised Manuscript Received January 28, 2010

Aortic heart valve disease is a growing health problem and a tissue-engineered aortic heart valve could be apromising therapy. In this paper, decellularized porcine aortic heart valve leaflets are used as scaffolds and loadedwith growth factor and heparin via layer-by-layer electrostatic deposition (LbL technique) with the final purposeto stimulate and control cellular processes. Binding and subsequent release of heparin and basic fibroblast growthfactor (bFGF) from aortic valve leaflets were assessed qualitatively by immunohistochemistry and quantitativelyby radioactive labeling methods. It was observed that the amount of heparin and bFGF bound to aortic heartvalve leaflets was directly proportional to the concentration of heparin and bFGF in the incubation medium.Release of heparin and bFGF from the decellularized heart valve leaflets at physiological conditions was sustainedover 4 days while preserving the biological activity of the released growth factor.

1. Introduction

The incidence of heart valve disease is a growing publichealth problem.1 Potential treatment involves replacement ofthe valve by a bioprosthetic valve, a homograft, or a mechanicalvalve prosthesis. However, currently available prosthetic valvesare all nonvital and lack the ability to grow, repair, and remodelin an in vivo environment in addition to several specificdisadvantages.2,3 A living, tissue-engineered, aortic heart valvewould overcome these limitations of current prostheses. For thispurpose, several strategies could be followed. One approach toproduce a tissue engineered aortic heart valve involves thepreseeding of a scaffold with cells, followed by an in vitro stageof cell growth and tissue formation. Subsequently, the constructis implanted in vivo and tissue growth and remodelling is furtherallowed to take place.4,5 In this work decellularized porcineaortic heart valves are used as biological scaffolds. These heartvalves have the advantage of enhancing cellular attachment andretaining many of the mechanical and structural properties suchas tensile strength and the unique extracellular matrix composi-tion of the aortic heart valve.6 During the decellularization ofthe porcine aortic heart valves, endothelial cells that cover thesurface of the valve, interstitial fibroblasts, smooth muscle cells,and myofibroblasts and proteoglycans are removed. Finally, amatrix consisting of merely collagen and elastin remains.7–11

Upon reseeding of a decellularized matrix, migration, pro-liferation, and if necessary transdifferentiation have to take placeto obtain a vital tissue. This is when growth factors come intoplay to stimulate and control cellular processes. Taking the shorthalf-life of growth factors into account, it would be beneficialto develop a system that could load high amounts of growthfactors combined with a controlled release of these growth

factors. Several techniques aiming to incorporate bioactivecomponents into tissue engineering matrices have been reported,including covalent as well as noncovalent coupling of growthfactors to scaffolds.12,13 Because covalent coupling of growthfactors can cause loss of biological activity of proteins,noncovalent binding of growth factors is preferred in this work.14

Basic fibroblast growth factor (bFGF) is a heparin-bindinggrowth factor belonging to the FGF family which stimulatesproliferation of fibroblasts.15 Heparin is a glycosaminoglycanwith a well-known in vivo behavior and will be used in thiswork to replace the proteoglycans, which were removed duringthe decellularization process. Because proteoglycans act to resistcompressive forces during valve opening and closure, they areimportant components of the heart valve.16 Moreover, gly-cosaminoglycans may function as a reservoir for growth factorsand they are involved in adhesion, migration, proliferation, anddifferentiation of cells.17–19 Heparin has many advantages incombination with bFGF or other growth factors and manygrowth factors are known as heparin-binding growth factors.Binding of bFGF to heparin induces a conformational changein the bFGF molecule resulting in an increased resistance againstthermal denaturation, enzymatic degradation, and a reducedinactivation at acidic pH.20–22 Yayon et al. suggested thatbinding of bFGF to heparin facilitates the binding of bFGF tohigh-affinity cell membrane receptors.23

In this paper, decellularized aortic heart valve leaflets areloaded with growth factors, using the layer-by-layer technique(LbL technique).24–27 This technique commonly involves thesequential adsorption of oppositely charged polymers (i.e.,polyelectrolytes) on a charged surface.28,29 The driving forcefor the formation of LbL films comes from the alternateovercompensation of the surface charge after each depositionof an oppositely charged polyelectrolyte. bFGF was chosen asthe positively charged polymer and heparin as the negativelycharged polymer. Several studies reported on the use of syntheticmatrices for incorporation and delivery of growth factors;

* To whom correspondence should be addressed. Phone: +32 9 264 8074. Fax: +32 9 222 82 36. E-mail: br.degeest@ugent.be.

† Department of Pharmaceutics.‡ Department of Molecular Biomedical Research.§ Department of Pharmaceutical Analysis.

Biomacromolecules 2010, 11, 1002–10081002

10.1021/bm9014649 2010 American Chemical SocietyPublished on Web 02/15/2010

however, the use of biological materials is rarely described.30–32

Here we aim to assess whether it is possible to perform thelayer-by-layer technique on decellularized aortic heart valveleaflets, while preserving the biological activity of the releasedgrowth factor.

2. Materials and Methods

2.1. Electrophoretic Mobility. Polystyrene beads (10 µm, sul-fonated; Microparticles GmbH) were coated with poly(ethyleneimine)(PEI; 22 kDa; Sigma-Aldrich), heparin (Diosynth Biotechnology), andbasic fibroblast growth factor (bFGF; PeproTech) in combination withbovine serum albumin (BSA; Sigma-Aldrich) or Tween 20 (Sigma-Aldrich). The electrophoretic mobility of the coated polystyrene beadswas measured using a Malvern Zetasizer 2000 (Malvern Instruments).

Adsorption of the polymers on the polystyrene beads was performedby the incubation of the beads during 30 min in the appropriatemacromolecule solution followed by two successive washing steps withwater. PEI, heparin, and bFGF were, respectively, used in a concentra-tion of 1 mg/mL, 1 mg/mL, and 1.5 µg/mL.

2.2. Cell Culture. Human dermal foreskin fibroblasts were obtainedfrom ATCC (CRL-2522). Cells were cultured in Eagle’s minimumessential medium (EMEM; ATCC), supplemented with streptomycin/penicillin, and 20% heat inactivated fetal bovine serum (FBS; Gibco)and grown at 37 °C in a humidified atmosphere containing 5% CO2.Experiments were performed at population doubling 30.

2.3. Matrix Preparation. Decellularization of the porcine aorticheart valves was performed using a patented detergent-enzymatictreatment.33,34 First, isolated porcine aortic heart valves were alterna-tively subjected to a mild alkaline hypotonic (pH 8) and hypertonicbuffered solution (pH 8), supplemented with phenylmethylsulfonylfluo-ride (PMSF), streptomycin/penicillin solution, and butylated hydroxya-nisole (BHA). Performing these steps resulted in the rupture of thenative cells and partially extracted cytoplasmic elements and solubleextracellular matrix. Second, to digest nucleic acids and cellularmembranes, the valves were treated enzymatically (DNase I, RNaseA, trypsin and phospholipases A2, C and D). Finally, the aortic heartvalves were washed in a magnesium and calcium-free chelating solution.All products were purchased from Sigma-Aldrich.

2.4. Collagen Determination of Aortic Heart Valve Leaflets. Thetotal collagen content of six lyophilized decellularized aortic valveleaflets was determined according to the protocol of The InternationalStandards Organisation; ISO/DIS 3496.2. The lyophilized decellularizedaortic valve leaflets were hydrolyzed using 6 M HCl at 110 °C during24 h to release hydroxyproline. Hydroxyproline was oxidized withchloramin-T which resulted in the formation of pyrrole-2-carboxylicacid and pyrrole. Pyrrole was determined using a color reaction withEhrlichs reagent (p-dimethylaminobenzaldehyde) in perchloric acid andisopropanol to give dipyrryl-phenylmethene salt and ms-tetra-(4-dimethyl-aminophenyl)-porphin. The latter was determined spectro-photometrically at 558 nm. To calculate the percentage of collagen, astandard curve of hydroxyproline solutions subjected to the colorreaction was used. A conversion factor of 8 was used.8

2.5. Scanning Electron Microscopy. Decellularized aortic heartvalve leaflets were dried on a silicone wafer followed by sputteringwith gold. SEM images were recorded with a Quanta 200 FEG FEIscanning electron microscope operated at an acceleration voltage of 5kV.

2.6. Aortic Valve Tissue Staining with Hematoxylin and Eosin.Decellularized aortic valve leaflets were dehydrated in ethanol and

embedded in paraffin. Sections of 5 µm were cut, stained withhematoxylin and eosin, and examined by light microscopy.

2.7. Immunohistochemistry of Heparin and bFGF Binding.2.7.1. Rhodamine Labeling of Heparin. Heparin was labeled withrhodamine isothiocyanate (RITC; Sigma-Aldrich) by slowly adding 12mg RITC in 60 mL of borate buffer (0.1 M; pH 8.5) to a vigorouslystirring solution of 300 mg heparin in 60 mL of borate buffer. After an

overnight reaction, the solution was dialyzed for 7 days against distilledwater with regular renewal of the water. Finally, RITC-heparin wasobtained as a purple powder after lyophilization. Coupling of heparinto rhodamine isothiocyanate occurred through amide bond formationbetween primary amines of heparin and isothiocyanate groups of RITC.

2.7.2. Visualization of Heparin Binding. Decellularized aortic heartvalve leaflets were incubated in a 1 mg/mL RITC-heparin in PBSsolution (Gibco). Two washing steps by soaking the aortic valve leafletsin excess PBS were performed to remove nonadsorbed RITC-heparin,and subsequently, the tissues were dehydrated in ethanol and embeddedin paraffin. Paraffin sections (5 µm) were cut with a microtome andvisualized with a Nikon EZ-C1 confocal microscope.

2.7.3. Visualization of bFGF Binding. RITC-heparin loaded aorticheart valve leaflets were incubated in aqueous medium containing 1µg/mL bFGF and 0.1% Tween 20 in an acetate buffer (50 mM; pH 5).Two washing steps by soaking the aortic heart valve leaflets in excessacetate buffer were performed to remove nonadsorbed bFGF, andsubsequently, the tissue was embedded in Tissue Tek (Sakura). Cryosections (10 µm) were cut with a cryo-microtome, washed in PBScontaining 1% BSA, and incubated overnight at 4 °C with a biotinylatedbFGF antibody (1/100 dilution of a 1 mg/mL stock in PBS containing1% BSA; PeproTech). After washing with PBS containing 1% BSA,fluorescent staining was performed with Cy5-labeled streptavidin (1/25 dilution of a 1 mg/mL stock solution; Zymed Laboratories) followedby washing with PBS. Microscopy images were recorded on a NikonEZ-C1 confocal microscope.

2.8. 123I and 125I Labeling of Heparin. Heparin was labeled with123I according to the Bolton Hunter method. Briefly, 100 mg heparinwas incubated during 3 h with a solution of water-soluble Bolton Hunter(5 mg/mL; Pierce) at pH 9 (borate buffer 0.1 M) in an ice bath, followedby an overnight dialysis of the reaction mixture against PBS (pH 7.4)to remove unbound Bolton Hunter reagent. Binding of the BoltonHunter by amide binding to heparin was possible due to the presenceof few primary amine groups in heparin.35 The heparin-Bolton Hunterwas incubated with 123I in a vial (coated with 70 µg iodogen) in PBSduring 20 min at room temperature. After the incubation period, residual123I was separated from the 123I-heparin by size exclusion chromatog-raphy on a PD-10 column. The concentration of the 123I-heparin solutionwas determined via size exclusion chromatography (PL aquagel-OH30 8 µm, 300 × 7.5 mm; Polymer laboratories, Church Stretton, U.K.)coupled with a spectrophotometer at a wavelength of 210 nm. Themobile phase was PBS (pH 7.4), the flow rate 1 mL/min. The obtained123I-heparin solution was further diluted in PBS. Labeling of heparinwith 125I was performed via a similar method.

123I-heparin was used to investigate the binding of the heparin todecellularized aortic heart valve leaflets, while 125I-heparin was usedto assess the release of heparin from decellularized aortic heart valveleaflets due to its longer half-life.

2.9. 123I and 125I Labeling of bFGF. bFGF was labeled with 123Iby the incubation of 50 µg bFGF with 123I in a vial coated with 70 µgiodogen in PBS containing 0.1% Tween 20 during 20 min at roomtemperature. After the incubation period, residual 123I was separatedfrom 123I-bFGF on a PD-10 column by size exclusion chromatographyusing acetate buffer (pH 5; 50 mM) containing 0.1% Tween 20 aseluent. The concentration of the radioactive bFGF was calculated viathe percentage of nonspecific adsorption of bFGF on the PD-10 columnand the original amount of bFGF, and the concentrated 123I-bFGFsolution was further diluted in acetate buffer (pH 5; 50 mM) containing0.1% Tween 20. Labeling of bFGF with 125I was performed via a similarmethod.

Similar to the iodine labeled heparin, 123I-bFGF was used toinvestigate the binding of the growth factor to decellularized aorticheart valve leaflets, while 125I-bFGF was used to assess the release ofbFGF from decellularized aortic heart valve leaflets.

2.10. Binding and Subsequent Release of Heparin from Decel-lularized Aortic Heart Valve Leaflets. Decellularized aortic heartvalve leaflets were incubated during 1 h under shaking conditions (400

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min-1) with 3 mL of heparin solution, followed by two successivewashing steps during 10 min in PBS. After deterioration of theradioactivity of 123I-heparin, the leaflets were lyophilized followed bythe assessment of their dry mass.

Release of heparin from the loaded heart valve leaflets was performedin physiological medium (PBS). The adsorbed and released iodinelabeled heparin was measured in a Packard cobra automated γ-counter.All experiments were performed in triplicate.

2.11. Binding and Subsequent Release of bFGF from Decellu-larized Aortic Heart Valve Leaflets. Decellularized aortic heart valveleaflets were incubated during 1 h under shaking conditions (400 min-1)with consecutively 3 mL of heparin solution (1 mg/mL) and 3 mL ofbFGF solution, each followed by two successive washing steps during10 min in PBS after heparin binding and in acetate buffer containing0.1% Tween 20 after bFGF binding. After deterioration of theradioactivity of 123I-bFGF, the leaflets were lyophilized followed bythe assessment of their dry mass.

Release of bFGF from the loaded heart leaflets was performed inphysiological medium (PBS containing 0.1% Tween 20). The adsorbedand released iodine labeled bFGF was measured in a Packard cobraautomated γ-counter. All experiments were performed in triplicate.

2.12. Preservation of the Biological Activity of the ReleasedbFGF. To investigate the preservation of the biological activity,decellularized aortic heart valve leaflets were loaded with heparin andheparin/bFGF, as described in sections 2.10 and 2.11 followed by theirrelease in EMEM containing 0.5% FBS. As controls, solutions ofheparin and heparin/bFGF of equal concentration as the release mediumwere used. Human dermal fibroblasts were seeded in a 96-well plateat a density of 2500 cells per well and incubated with the test medium.After 1, 3, and 5 days, the cells were quantified using a p-nitrophenylphosphate (pNPP; Sigma-Aldrich) cell viability assay. Cells wereincubated with a pNPP solution under acidic conditions during 2 h at37 °C and 5% CO2. During this incubation period, p-nitrophenylphosphate was hydrolyzed by acid phosphatase resulting in the releaseof p-nitrophenol and phosphate. The reaction was stopped with NaOH,resulting in the formation of a yellow color, which was spectrophoto-metrically measured at 405 nm. All experiments were performed intriplicate.

2.13. Statistical Analysis. Descriptive statistics are expressed asmean ( standard deviation. Data regarding preservation of thebiological activity of the released bFGF were analyzed via 3-wayANOVA. p-Values < 0.05 are considered significant.

3. Results and Discussion

3.1. Electrophoretic Mobility. Growth factors are oftenprovided in combination with bovine serum albumin (BSA) ascryoprotectant during lyophilization and to avoid nonspecificadsorption to the surface of the recipient upon reconstitution inaqueous medium. Taking into account the pKa values of 9.5936

for bFGF and 4.937 for BSA it is reasonable to assume that atphysiological conditions (i.e., pH 7.4; 150 mM NaCl) electro-static complexes will be formed in solution between bFGF and

BSA. This will hamper the use of electrostatic forces toincorporate bFGF as such in an electrostatically assembledmultilayer film (further denoted as LbL film) onto a solidsurface. Therefore, we initially investigated the parametersinvolved in LbL build-up using polystyrene beads as templateand electrophoretic mobility measurements as detection tech-nique. To avoid nonspecific adsorption of bFGF, Tween 20 wasused as an alternative for BSA.38 Tween 20 is a nonionicblockcopolymer surfactant and in presence of bFGF, electrostaticcomplexation in solution between Tween 20 and bFGF will beavoided, allowing the growth factor to participate in theformation of a LbL film.

Electrophoretic mobility measurements showed the polysty-rene beads to exhibit a γ-potential of -50 ( 1.89 mV. To mimicthe surface charge of an aortic heart valve leaflet, exhibiting apositive surface charge due to the presence of collagen, thepolystyrene beads were coated with a layer of polyethyleneimine(PEI), which is commonly used in LbL assemblies as adhesionpromoter in a first layer, resulting in a positive γ-potential of18 ( 2.58 mV. Upon deposition of a heparin layer chargereversal took place and a γ-potential of -48 ( 3.37 mV wasmeasured. In a next step, the beads were incubated with asolution of 1.5 µg/mL bFGF combined with respectively 0.1%BSA or 0.1% Tween 20, resulting in a γ-potential of -33.2 (5.76 mV, respectively, -12.6 ( 3.07 mV, indicating that,compared to the presence of BSA, the presence of Tween 20allowed the γ-potential to increase more pronounced upondeposition of bFGF (Figure 1). As a control (data not shown),PEI/heparin coated beads were incubated with a 0.1% BSA anda 0.1% Tween 20 solution, resulting in respectively a γ-potentialof -31.8 ( 4.83 mV and -31.9 ( 3.91 mV. Another set ofcontrols (data not shown) included the incubation of the PEIand heparin-coated beads with a 1.5 µg/mL solution of bFGFin a polypropylene vial precoated with BSA or Tween 20, whichresulted in respectively a γ-potential of -26.7 ( 3.82 mV and-15.6 ( 3.2 mV.

In all cases, adsorption of bFGF resulted in an increase ofthe γ-potential due to the positive charge of the growth factorat physiological conditions. The highest increase of the γ-po-tential was obtained when the beads were incubated with a 1.5µg/mL solution of bFGF containing Tween 20 or when the beadswere incubated with a 1.5 µg/mL solution of bFGF in a vialwhich was precoated with Tween 20. These results indicate anideal formation of a LbL film using bFGF in combination withTween 20. All the experiments in this paper, including theformation of LbL film on the heart valve leaflets, wereperformed using bFGF in combination with 0.1% Tween 20.

3.2. Characterization of the Aortic Heart Valve Leaflets.3.2.1. Collagen Content of the Aortic Heart ValVe Leaflets.Collagen fibers provide most of the mechanical and tensilestrength of heart valves.10 The collagen content of decellularized

Figure 1. Evolution of the zeta-potential (mV) after the subsequent deposition steps in LbL coating of polystyrene beads.

1004 Biomacromolecules, Vol. 11, No. 4, 2010 De Cock et al.

aortic heart valve leaflets was determined according to theprotocol of The International Standards Organization; ISO/DIS3496.2 considering the mass of the lyophilized matrices andwas measured to be 85% ((4%). This result is consistent withliterature describing the major substance of the aortic heart valvebeing collagen.11 Studies reported on the predominant formsof collagen found in the valves being collagen type I (74%),type III (24%), and only a small amount of type V.10 Theremaining part may be elastin.10

3.2.2. Scanning Electron Microscopy and H&E Staining. Thestructure of the decellularized aortic heart valve leaflets wasanalyzed via hematoxylin and eosin staining of microtomeddecellularized heart valve leaflets and scanning electron mi-croscopy of whole decellularized heart valve leaflets (Figure2). These results show the presence of small pores (pore sizevaries from approximately 1-15 µm) in the tissue and on thesurface of the leaflet. In Figure 2D the fibers of the decellularizedaortic heart valve leaflet can be clearly observed.

3.3. Layer-by-Layer Deposition of Heparin and bFGFon Decellularized Aortic Heart Valve Leaflets.3.3.1. QualitatiVe Analysis by Immunohistochemistry. The LbLfilm on the decellularized aortic heart valve leaflets consistedof heparin (Figure 3), which is negatively charged, and bFGF,which is positively charged at physiological conditions (i.e pH7.4). Heparin was red fluorescent labeled with rhodamine andincubated with aortic heart valve leaflets at a concentration of1 mg/mL heparin. Subsequently, the heart valve leaflets wererinsed with PBS to remove nonadsorbed heparin followed byincubation of the heart valve leaflets in a 1 µg/mL bFGF

solution. After the incubation period and washing steps,microtome sections were made of the leaflet followed byimmunohistochemical staining with bFGF antibody. Figure 4shows confocal microscopy images of stained heart valveleaflets. The rhodamine-labeled (red fluorescent) heparin wasvisible as small stripes presumably along the collagen fibers.Although the binding of heparin to the tissue in the cryosectionswas homogeneously distributed throughout the heart valveleaflet, we saw in the paraffin sections that binding of heparinwas higher to the surface of the tissue compared to the bindingdeeper in the tissue. The antibody staining against bFGF wasassigned the blue color, as both rhodamine and Cy5 areconsidered red fluorophores, however, with a sufficient wave-length gap to record them separately. The bFGF was apparentlyalso present in a stripe-like pattern, similar to that of heparin.Control experiments with heart valve leaflets without heparincoating yielded much lower signal of bFGF (data not shown).For this reason binding of bFGF to leaflets without heparincoating was not studied in the following experiments. Takentogether, these images indicate that both heparin and bFGF arewell adsorbed to the decellularized heart valve leaflets.

Binding of heparin and bFGF to the decellularized aortic heartvalve leaflet can occur through electrostatic interaction, althoughother binding mechanisms may be involved as well. Decellu-larized aortic heart valve leaflets contain collagen and elastinas major components. Heparin can interact through electrostaticinteraction with collagen type I through the positively chargedgroups. It is also described in literature that collagen containsa high-affinity receptor for heparin explaining the high affinitybetween heparin and collagen type I.39–41 Interaction betweenheparin and bFGF can be explained by ionic interaction betweenboth 2-O-sulfate groups and the N-sulfate group of heparinmolecules and certain lysine and arginine residues in bFGF.30

3.3.2. QuantitatiVe Analysis by RadioactiVe Labeling. De-cellularized aortic heart valve leaflets were incubated withradioactive labeled heparin (123I-heparin) within a concentrationrange from 0 to 5.073 mg/mL. Another set of experimentsincluded incubation of decellularized aortic heart valve leafletswith heparin (1 mg/mL) followed by incubation with radioactivelabeled bFGF (123I-bFGF) within a concentration range from 0to 1.764 µg/mL. A concentration of 1 mg/mL of heparin wasused as it is a commonly used concentration to construct LbLfilms. bFGF was used within the µg/mL range because theeffective concentration of this growth factor is within the ng/mL range. Previously, Somers et al. used the wet weight of thedecellularized aortic heart valve leaflets to express the amountof bound heparin and bFGF to the tissue.32 Because the wetweight of the leaflets is characterized by a high variability (datanot shown), using this data to express the amount of boundpolymer to the decellularized aortic heart valve leaflets is merelyan approximation. In this work, we used the mass of thelyophilized aortic heart valve leaflets to express the amount ofheparin and bFGF bound to the leaflets. Figure 5 demonstratesthat the amount of bound heparin (Figure 5A) and bFGF (Figure5B) is directly proportional to the concentration of the mediumin which the heart valve leaflets were incubated. However, onehas to keep in mind that these curves are probably the first partof a curve leading to a plateau indicating saturation of thebinding of heparin and bFGF to the decellularized aortic heartvalve leaflets.42

3.3.3. Release of Heparin and bFGF from the Decellular-ized Aortic Heart ValVe Leaflets. Decellularized aortic heartvalve leaflets were loaded with 125I-heparin in a 1 and 4 mg/mL solution and with 125I-bFGF in a 0.316 and 1.265 µg/mL

Figure 2. (A,B) Light microscopy images at different magnificationof paraffin sections of decellularized aortic heart valve leaflets stainedwith hematoxylin and eosin. (C,D) Scanning electron microscopyimages at different magnification of decellularized aortic heart valveleaflets.

Figure 3. Molecular structure of heparin in its ionized form.

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solution. Release of heparin and bFGF, evaluated at physiologi-cal conditions via 125I labeling of the polymers due to the longerhalf-life of 125I compared to 123I, was mainly driven bycompetition with salt ions. For both components, the releaseprofile is characterized by an initial burst release, followed bya sustained release over 4 days. The initial burst release can beexplained by the release of material which was loosely boundnear the surface of the leaflets. The sustained release can beexplained by the release of polymer, which was located moredeeply into the structure of the leaflets.39 After an incubationperiod at physiological conditions of 4 days 70% of theoriginally bound heparin and 80% of the originally bound bFGFhas been released (Figure 6).

3.4. Preservation of the Biological Activity of the Re-leased bFGF. An important issue regarding binding and releaseof bFGF from decellularized aortic heart valve leaflets is thepreservation of the biological activity of the released growthfactor. As bFGF stimulates proliferation of human dermalfibroblasts,15 we performed a comparative cell proliferationstudy between on one hand freshly prepared heparin, heparin/bFGF solutions, and on the other hand heparin, heparin/bFGF,

released from the aortic heart valve leaflets. Aortic heart valveleaflets loaded with respectively heparin (1 mg/mL) or heparin(1 mg/mL)/bFGF (1 µg/mL) were incubated for 4 days inEMEM containing 0.5% FBS. From the binding data (Figure5) one can calculate the amount of adsorbed heparin (i.e., 5.3µg/mg tissue) and bFGF (25.5 ng/mg tissue). As the releaseafter 4 days at physiological conditions of heparin and bFGFis, respectively, 70 and 80% (Figure 6), the concentration ofheparin and bFGF in the release medium can be calculatedconsidering the mass of the leaflets. These solutions were dilutedin cell medium to obtain a bFGF concentration of 10 ng/mL,which should according to literature be optimal for cellproliferation.15

Cell proliferation was assessed by culturing human dermalfibroblasts in the presence of the diluted release medium,followed by analysis of the cell viability with a common pNPPassay after, respectively, 1, 3, and 5 days. Figure 7 shows thecomparative results between heparin and heparin/bFGF releasedfrom aortic heart valve leaflets and freshly prepared heparin andheparin/bFGF solutions of equal concentrations as calculatedfor the released heparin and heparin/bFGF. The cell viability

Figure 4. (A) Confocal microscopy images of paraffin sections showing a decellularized aortic heart valve leaflet incubated with heparin (A1),a transmission image (A2), and an overlay (A3). (B) Confocal microscopy images of cryosections showing a decellularized aortic heart valveleaflet incubated with respectively heparin (B1) and bFGF (B2) and an overlay of the heparin and bFGF binding (B3). The red fluorescence isdue to the heparin labeled with rhodamine, the blue fluorescence is due to the immunohistochemical staining with bFGF antibody and CY5-labeled streptavidin.

Figure 5. Binding of (A) heparin to decellularized aortic heart valve leaflets and (B) bFGF to heparin loaded decellularized aortic heart valveleaflets as a function of the heparin, respectively, bFGF concentration in the incubation medium.

1006 Biomacromolecules, Vol. 11, No. 4, 2010 De Cock et al.

was expressed as a percentage relative to cells cultured inheparin- and bFGF-free medium as control.

These series of experiments demonstrated that heparin didnot influence cell proliferation, as within the heparin group nostatistical significant difference between days 1, 3, and 5 wasobserved. In contrast, bFGF did stimulate cell proliferation asrelative cell numbers increased as a function of time. Withinthe group of heparin/bFGF, statistical significant differencesbetween days 1 and 3, days 1 and 5, and days 3 and 5 wereobserved. These findings suggested that the activity of thereleased bFGF was preserved after being bound to an aorticheart valve leaflet and subsequently being released.

4. Conclusions

In this work, we performed the layer-by-layer technique ondecellularized aortic heart valve leaflets as biological matrices.Binding of heparin and bFGF to aortic valve leaflets was shownfollowed by release of bFGF and heparin while preserving theirbiological activity. We also demonstrated that, to incorporategrowth factors within a multilayer film, it is preferable tocombine the growth factor with Tween 20 instead of BSA toavoid nonspecific adsorption of bFGF.

Human dermal mesenchymal fibroblasts are an attractivesource of progenitor cells for myofibroblasts, making them aninteresting cell type for the repopulation of the decellularizedaortic heart valve leaflets.15 In future research we will focus onthe interaction between heparin/growth factor-loaded heart valveleaflets with human dermal mesenchymal fibroblasts. So far we

were not yet able to demonstrate repopulation of heparin/bFGF-loaded heart valve leaflets. This is likely due to the limited poresize of the leaflets that does not allow spontaneous inwardmigration of human dermal mesenchymal fibroblasts. On theother hand, improvement could be obtained when using anoptimal growth factor cocktail, for example, incorporated withina polyelectrolyte multilayer film, comprising of growth factorssuch as bFGF, transforming growth factor beta1 (TGF-�1), andepidermal growth factor (EGF), as reported by Narine et al., tostimulate proliferation, transdifferentiation, and migration of invitro cultured human dermal mesenchymal fibroblasts.15 Theseinvestigations are currently ongoing.

Acknowledgment. L.J.D.C. wishes to express her gratitudeto the Institute for the Promotion of Innovation by Science andTechnology in Flanders (IWT-Flanders) for their financialsupport. B.G.D.G. acknowledges the FWO for a postdoctoralscholarship.

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Figure 6. Cumulative release curves of (A) heparin and (B) bFGF from the decellularized aortic heart valve leaflets in physiological medium.Curves were recorded for two concentrations of heparin, respectively, bFGF in the loading medium.

Figure 7. Comparative cell viability histograms of human dermal fibroblasts incubated for 1, 3, and 5 days with (black bars) heparin and heparin/bFGF released from aortic heart valve leaflets, respectively, (white bars) heparin and heparin/bFGF solutions of equal concentrations as therelease media. The concentration of bFGF was 10 ng/mL (*p < 0.05).

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