Feasibility of pig and human-derived aortic valve interstitial cells seeding on fixative-free decellularized animal pericardium Rosaria Santoro,1* Filippo Consolo,2* Marco Spiccia,2 Marco Piola,2 Samer Kassem,3 Francesca Prandi,1 Maria Cristina Vinci,1 Elisa Forti,1 Gianluca Polvani,4 Gianfranco Beniamino Fiore,2 Monica Soncini,2* Maurizio Pesce1* 1

 di Ingegneria Tissutale, Centro Cardiologico Monzino, IRCCS, Milan, Italy Unita Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milano, Italy 3 Divisione di Cardiochirurgia, Centro Cardiologico Monzino, IRCCS, Milan, Italy 4 , Sezione cardiovascolare, Universita  di Milano, Milan, Italy Dipartimento di Scienze Cliniche e di Comunita 2

Received 28 July 2014; revised 9 February 2015; accepted 16 February 2015 Published online 23 March 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33404 Abstract: Glutaraldehyde-fixed pericardium of animal origin is the elective material for the fabrication of bio-prosthetic valves for surgical replacement of insufficient/stenotic cardiac valves. However, the pericardial tissue employed to this aim undergoes severe calcification due to chronic inflammation resulting from a non-complete immunological compatibility of the animal-derived pericardial tissue resulting from failure to remove animal-derived xeno-antigens. In the mid/longterm, this leads to structural deterioration, mechanical failure, and prosthesis leaflets rupture, with consequent need for reintervention. In the search for novel procedures to maximize biological compatibility of the pericardial tissue into immunocompetent background, we have recently devised a procedure to decellularize the human pericardium as an alternative to fixation with aldehydes. In the present contribution, we used this procedure to derive sheets of decellularized pig pericardium. The decellularized tissue was first tested for the

presence of 1,3 a-galactose (aGal), one of the main xenoantigens involved in prosthetic valve rejection, as well as for mechanical tensile behavior and distensibility, and finally seeded with pig- and human-derived aortic valve interstitial cells. We demonstrate that the decellularization procedure removed the aGAL antigen, maintained the mechanical characteristics of the native pig pericardium, and ensured an efficient surface colonization of the tissue by animal- and human-derived aortic valve interstitial cells. This establishes, for the first time, the feasibility of fixative-free pericardial tissue seeding with valve competent cells for derivation of tisC 2015 Wiley Periodicals, Inc. sue engineered heart valve leaflets. V J Biomed Mater Res Part B: Appl Biomater, 104B: 345–356, 2016.

Key Words: animal-derived pericardium, decellularization, aortic valve interstitial cells, mechanical properties, cell seeding

How to cite this article: Santoro R, Consolo F, Spiccia M, Piola M, Kassem S, Prandi F, Vinci MC, Forti E, Polvani G, Fiore GB, Soncini M, Pesce M. 2016. Feasibility of pig and human-derived aortic valve interstitial cells seeding on fixative-free decellularized animal pericardium. J Biomed Mater Res Part B 2016:104B:345–356.

INTRODUCTION

The pericardial membrane of animal origin (mainly porcine and bovine) treated with glutaraldehyde (GA) is the election material employed in the manufacture of commercial bioprosthetic valves. While the use of this tissue in heterograft valves fabrication dates to >50 years ago,1,2 the transplantation of these devices in humans still provides severe side effects resulting from the lack of a complete biological compatibility. Apart from mechanical failures, which may result from shortcomings in the production or the surgical implantation procedures, structural leaflet deterioration is still the principal cause of bio-prosthetic valve failure in the mid/ long term, affecting significant portions of the patients populations, especially in the young age.3 Deterioration of the

implants is caused primarily by a chronic inflammatory condition resulting from a non-complete detoxification of the fixative remnants from in the pericardium,4,5 or by the failure of the fixation protocols to remove major xeno-antigens such as the 1,3 a-Galactose6–10 (a-Gal). Optimization of fixation procedures such as the setting of the most appropriate GA treatment times or the fixative dilution,11 detoxification of aldehyde residues by enzymatic treatments and nanocoating,12 aldehyde residues extraction by solvent incubation13 or the manufacture of pericardiumderived ECM-residues coated scaffolds14 have been proposed. On the other hand, it is evident that the leaflet preparation method for the “ideal” valve prostheses still does not exist,15 leaving space for further basic research.

*These authors contributed equally to this work. Correspondence to: M. Pesce; e-mail: [email protected] Contract grant sponsor: Ricerca Corrente

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Alternative modalities for leaflet fabrication, based on pericardium decellularization have been proposed. These methods, originally used to decellularize porcine valves,16,17 are not based on GA fixation but on osmotic lysis of the cells populating the tissue, followed by extraction of cell remnants with ionic/nonionic detergents, and treatment with enzymes to remove the genetic material (DNA/RNA). Although fixation-free cell removal procedures have been proven successful for the lower inflammatory potential of the decellularized grafts in experimental models,18 and have been employed to perform decellularization of humanderived pericardium for potential employment as an homograft tissue in surgery,19,20 or the production of scaffolds for the derivation of tissue engineered heart valve (TEHV) leaflets,21,22 they are not free of contraindications. In fact, cases of decellularized valve implant rejection have been reported in children due to strong immune and foreign body reactions.23,24 In the present investigation we adopted a fixation-free decellularization method previously set up by us,21 to obtain cell-free pig pericardium patches. After decellularization, the tissue was antigen and mechanically characterized, and thereafter used as a substrate for the seeding of pigand human-derived aortic valve interstitial cells (aVICs). We show the feasibility of aVICs seeding onto fixative-free decellularized pig pericardium patches as a first step toward the derivation of “off-the-shelf” TEHVs containing natural valve-resident cells. MATERIALS AND METHODS

Derivation of pericardium and decellularization procedure Pig hearts (6–9 months of age) were obtained at the local slaughterhouse. Parietal pericardial tissue was dissected from the left ventricle and then treated as described in our previous work.21 This procedure is a modification of the protocol originally used by others19,20 and consists in a sequential incubation of the pericardial tissue into (i) a hypotonic buffer (10 mM Tris-HCl; pH 8.0), (ii) a detergent hypotonic solution containing 0.1% (w/v) sodium dodecylsulfate (SDS), and (iii) a nucleic acid removal solution containing 50 U mL21 deoxyribonuclease-I and 1 U mL21 ribonuclease-A. After decellularization, the pericardial tissue was either fixed for histological sectioning, staining and immunofluorescence, or prepared for mechanical testing or, finally, decontaminated for cell seeding. Mechanical properties of the decellularized pericardium were compared with those of GA fixed specimens, obtained by incubation for 48 h in a PBS (pH 7.4)-buffered solution containing 0.6% GA,10,25,26 followed by a 48 h rinsing step in bi-distilled water, and further washing in PBS containing 2% Penicillin/ Streptomicin for 3 days. Histological sectioning, staining, and immunofluorescence Pericardium (before and after decellularization) was fixed in formalin and embedded in paraffin. Histological sections (4– 6 lm) were cut and subsequently dewaxed and stained

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with Masson’s trichrome. The presence of a-Gal epitope (Gala1-3Gal b1-4GlcNAc-R) was assessed by immunofluorescence analysis. The monoclonal antibody M86 (Enzo Life Sciences) was used as specific a-Gal detector. Briefly, after antigen retrieval the sections were stained with the M86 antibody (1:50) for 1 h at room temperature followed by recognition with a secondary antibody (donkey anti-mouse AlexaFluor 488, Life Technologies). Sections were then counterstained with DAPI (Sigma–Aldrich). Images were acquired with Axiovert/Apotome optical/fluorescence microscopes (Zeiss). Uniaxial tensile lading tests Uniaxial tensile loading (UTL) tests were performed on native (N), decellularized (DE) and GA-fixed (GA) porcine pericardial samples to reveal any potential alteration of the tissue biomechanical characteristics induced by decellularization and/or GA-fixation procedures with respect to native pericardium. Before testing, all the samples were maintained in PBS at 4 C. Dog-bone shaped (4-mm width and 20-mm height) specimens were dissected from pericardial tissue sheets and no preferential orientation was considered when cutting the specimens, assuming porcine pericardium isotropy.27 The ends of the specimens were glued with cyanoacrylate adhesive in between of frames made of finegrained emery article to facilitate uniform gripping and to avoid sample slippage. Then the specimens were mounted onto the clamps of the testing machine (MTS hydraulic testing machine equipped with 100-N loading cell, MTS System Corporation) and the sides of the sandpaper frame were cut. This procedure is described in Ref. 28 and was already employed by us for human-derived pericardium.21 Tissue specimens were preloaded up to 0.01N and preconditioned through loading-unloading cycles (ranging from 6 to 8 cycles) at 15% maximum strain with an elongation rate of 10 mm min21, until the loading-unloading curves were almost superimposed. After tissue preconditioning, the specimens were preloaded up to 0.01N. The thickness of each specimen was measured in three different positions along the specimen height by means of a digital caliper (CD15CPX, Mitutoyo Italiana S.r.l.) and the thickness mean value (tav) was used for mechanical parameters’ calculation. Also the specimen initial length was measured, and UTL test was conducted, at a constant velocity of 10 mm min21, until specimen failure. UTL tests were performed at room temperature and the specimens were maintained hydrated with PBS for the whole test duration. The stress–strain (r–E) behavior of each specimen of the three groups was acquired and the biomechanical properties of the pericardial tissue described by means of six parameters21,29: the elastic modulus (that is, the r–E curve slope) at low (Elow) and high (Ehigh) strain, representative of the tissue resistance due to the contribution of the elastin and collagen fibers composing the ECM, respectively; the tensile stress (rtrans) and strain (Etrans) values at the transition between the elastin and collagen stress–strain curve slopes; the maximum tensile stress (rmax) and strain (Emax) characterizing the failure phase of the specimen.

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FIGURE 1. (A) Schematics of the experimental setup developed for P–V curve analysis; in the zoomed-in area, a 3D CAD of the PDMS cartridge hosting the pericardial tissue patch is shown. (B) The elements composing the perfusion chamber. (C) The steps required to house and fasten the pericardial patch within the cartridge are depicted: the o-ring avoids tissue slippage during the test, ensuring confined flow-perfusion of the specimens.

Distensibility and permeability test An experimental setup was developed to evaluate and compare tissue distensibility and permeability properties of N, DE, and GA porcine pericardial tissues. The experimental layout (Figure 1) consists of: (i) a biocompatible and elastomeric silicone-made cartridge (polydimethylsiloxane, PDMS, Sylgard 184 Dow Corning Corporation) hosting roundshaped tissue specimens [Figure 1(A,B)], (ii) a nitrile butadiene rubber o-ring used to fasten the tissue within the cartridge, avoiding tissue slippage and ensuring confined-flow perfusion on a 8-mm-diameter tissue surface [Figure 1(B)]; (iii) a perfusion chamber adapted a previous study30 and composed of a housing, where the cartridge is hosted, and a reservoir containing the fluid (ddH2O) circulated during the test [Figure 1(A,B)]; (iv) a peristaltic pump (Ismatec IPC-N, IDEX Health & Science GmbH) delivering the fluid flow from the reservoir to the tissue specimen [Figure 1(A)]; (v) a pressure sensor (Press-S-000, Pendotech) [Figure 1(A)], and (vi) a I/O data acquisition system [Figure 1(A)] controlled by a custom-made software interface (DAQ-card USB-6211 and LabViewTM, National Instruments) developed for the real-time monitoring and acquisition of the pressure (P) acting on the perfused tissue patch in response to continuous fluid volume (V) infusion driven by the pump. Roundshaped tissue specimens were manually retrieved from pericardial tissue sheets (maintained in PBS at 4 C until the

test) using surgical scissors and housed within the PDMS cartridge, as described in Figure 1(C). For distensibility analysis, P–V curves were acquired at 10-lL min21 flow rate (Q). Tests were made in order to evaluate the effect of decellularization and GA fixation on passive distensibility properties of the pericardium. Within the perfusion chamber, the circular patches were subjected to biaxial inflation,31,32 that is, tretched in radial and circumferential directions, grossly mimicking the natural mechanical stimulus in the pericardium sac. This was done by expanding the pericardium patches with a known fluid volume, thus allowing to measure the changes in transmural pressure, and to estimate the pericardium distensibility properties. Four parameters descriptive of the tissue distensibility properties were extracted from P–V curves, as previously described33: the P–V curve slope at low (P’low) and high (P’high) infused volume values, representative of the tissue distensibility due to the contribution of the elastin and collagen fibers composing the ECM, respectively, and the pressure (Ptrans) and volume (Vtrans) values at the transition between the two characteristic P–V curve slopes (that is, at the transition between the elastin and collagen curve slopes). For permeability tests, P–V curves were acquired at 10 (Q10), 20 (Q10) and 30 (Q30) lL min21 and the plateau of the P–V curves (Pplateau) representative for effective fluid filtration, was identified to calculate the tissue fluid filtration coefficient

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(/), according to Darcy formulation, through linear regression analysis of the Q–Pplateau data points. Derivation, culture, and characterization of pig- and human-derived aortic valve interstitial cells Failing human aortic valves were obtained as discharged material after surgical valve replacement procedure. An informed consent approved by the Local Ethical Committee was sought and obtained to this aim. The non-calcific portions of explanted valves were processed by enzymatic treatment for aVICs extraction, using a protocol adapted from what described by Taylor et al.34 Briefly, incubation for 5 min on shaker at 37 C in Collagenase Type II (Worthington) solution (1000 U mL21), was performed to remove the endothelial layer. A second 2-h incubation under the same conditions allowed to isolate the aVICs, that were then plated for ex vivo amplification on 1% gelatin coated plastic cell culture dishes (10 cm diameter). Pig-derived aVICs were obtained using aortic valves of pigs of 6–9 months of age (see before) with the same procedure described above for the human cells. Cells from both species were grown and maintained in the following complete media: DMEM (Lonza) containing 150 U mL21 penicillin/streptomycin (Sigma–Aldrich) and 2 mM L-glutamine (Sigma–Aldrich), and 10% bovine serum (HyClone, Thermo Scientific). Cells used in this work were expanded for 4 passages after characterization of their basic growth characteristics (doubling time at passage 1: 7.6 6 3.6 days (n 5 17); doubling time at passage 4: 5.5 6 2.5 days (n 5 4)). Phenotype assessment of human aVICs isolated from 3 donors (72 6 5 years) was performed at passage 3 by flow cytometry as follows: cells were detached from tissue culture dishes with nonenzymatic dissociation buffer (Sigma–Aldrich) and stained to detect HLA-ABC, HLA-DR, CD31, CD44, CD73, CD90, and CD105 expression using PE-, APC- and FITC-conjugated mouse anti-human antibodies (BD Pharmigen). Analysis was performed using FACSCalibur and FACSAria (Beckton Dickinson) flow cytometers. Immunofluorescence to assess expression of Vimentin and aSMA markers was performed using the primary antibodies mouse anti human Smooth Muscle Actin (M0851-DAKO) and rabbit anti human Vimentin (5741-Cell Signaling). This was followed by incubation with secondary antibodies: Alexa 488 donkey anti mouse (Invitrogen) and Alexa 633 goat anti rabbit (Life Technologies). Pericardial patches preparation, decontamination, and cell seeding Following dissection from the heart, pericardial tissue was sectioned into 15 3 15 mm2 samples. Decellularization was then performed as described above, and followed by a decontamination procedure, consisting in 72-h incubation at 4 C in BASE 128 (AL.CHI.MIA S.r.l.), an EC certified decontamination medium containing an antibiotic/antifungal mixture (Gentamicin, Vancomycin, Cefotaxime and Amphotericin B) and approved for employment in Tissue Banking.35 Pericardial patches were fixed by a nitrile butadiene rubber o-ring into ad hoc manufactured holders, consisting in a

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PDMS cartridge connected to a polyoxymethylene conicshaped reservoir (1.5 mL volume), leaving 8-mm-diameter area available for cell seeding. All the components were sterilized by autoclave before use; however, in order to ensure the patch sterility after the manual assembly procedures, the reservoir was filled with BASE 128 and the tissue was incubated for 24 h at 4 C. Abundant washes in PBS were performed before aVICs seeding on the samples mounted on the PDMS cartridge. To optimize cell seeding, a two-steps protocol was performed: cells suspended in a small volume (0.5 mL) of complete media were deposited on the pericardium; after 2 h the medium volume was filled up to 1.5 mL. Experiments were performed comparing two seeding densities (low: 0.6 3 103 cell mm22, high: 3 3 103 cell mm22) and cells were cultured on the serosa (S) and fibrosa (F) surfaces of pericardium patches (see results) up to 72 h. RESULTS

Fixative-free decellularization procedure ensures complete genetic and xenoantigen removal without compromising pericardium ECM structure Initial tests were dedicated to verify the efficiency of the decellularization procedure to remove the cells from the porcine pericardium. This was performed by histological sectioning and observing under the light/fluorescence microscope after histochemistry and staining with nuclear dye DAPI. As already shown in the human pericardium,21 the structure and the composition of the tissue was not grossly affected by the osmotic treatment [Figure 2(A)]. In addition, both the Masson’s trichrome [Figure 2(A)] and the fluorescent nuclear staining with DAPI [Figure 2(B), left] confirmed the absence of cells and a complete removal of DNA. To assess whether the decellularization method was efficient in removing one of the main xenoantigens in prosthetic valve inflammation, an immunofluorecence using an antibody against the 1,3-aGal, was performed [Figure 2(B); right]; the absence of any detectable fluorescence in DE pericardium suggested the complete removal of this antigen. Structure characterization and aGal staining of the GAtreated tissue was not performed, as this has been already extensively done in previous studies, also including aGal detection in commercial pericardium-made valve bioprostheses.6–8,10,36,37 As shown in Figure 2(C), finally, the fixative-free decellularization procedure did not cause major variations of the tissue thickness compared with a conventional treatment with 0.6% GA. Mechanical and permeability testing of the fixative-free decellularized tissue The suitability of the decellularized pericardium as a material for the manufacture of bioprosthetic valves depends on the maintenance of its resistance to mechanical solicitations. For this reason, treatment with fixatives has been historically employed with the aim of maintaining the tissue mechanical characteristics.38–40 The representative mechanical behavior of the N, DE and GA pericardium was then assessed by UTL tests; results are reported in Figure 3(A),

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parameters obtained by UTL tests, except for rmax [Figure 3(C)]. In contrast, GA samples showed an overall increased elongation properties and higher stiffness, particularly evident in the low strain region [Figure 3(C)]. These results were confirmed by P–V curve analysis [distensibility tests, Figure 4(A–D)], showing that the DE pericardium was characterized by comparable distensibility properties to those of the untreated tissue [Figure 4(D)], thus reflecting a physiological-like behavior. P–V tests also suggested an overall increased stiffness of the elastin and the collagen fibers of the GA pericardium, as indicated by the significantly higher curve slopes (P’low, P’high), likely determined by treatment with GA. Finally, consistently with the altered behavior of the elastin fibers observed in the GA-treated group, the transition from the low to the high curve slope region was shifted toward higher volume and pressure values, as shown by a significantly higher Vtrans and Ptrans with respect to N and DE samples. The P–V curves of DE specimens also revealed a different trend in the final curve region; in fact, DE pericardium did not respond to a sudden pressure rise as N and GA tissues, but exhibited a “quasi-plateau” behavior [Figure 4(A,C)]. This suggested filtration of the infused fluid, likely due to an increased permeability of DE tissue, as a consequence of the massive cell removal [Figure 4(C) reported representative P–V curves acquired for DE tissue at different Q values]. Accordingly, a filtration coefficient for DE pericardium was calculated, equal to /DE 5 0.12 6 0.01 lL min21 mmHg21, while null / was assumed for N and GA tissue specimens, since no plateau behavior was observed for those groups. Taken together, our results show that the fixative-free decellularization procedure is conservative with respect to pericardium biomechanical properties but induces tissue fluid filtration.

FIGURE 2. (A) Masson’s thrichrome staining of transversally cut sections of the pig pericardium before (left) and after (right) decellularization procedure. S and F indicate the serosa and the fibrosa surfaces, respectively. Note the integrity of the collagen bundles and an evident thickness decrease. (B) Staining with nuclear dye DAPI (Blue fluorescence) and an antibody recognizing the a-GAL epitope (green fluorescence) in native (upper) and decellularized (lower) pericardium. The absence of any detectable fluorescence in both fluorescence channels acquired with identical exposure time shows the complete removal of genetic material and the pig xenoantigen. S and F indicate the serosa and the fibrosa surfaces, respectively while V shows two vessels in the fibrosa side of the tissue. (C) Thickness measurement showed a substantial thickening of the tissue following the decellularization procedure. In this test, the fixation with GA was also included as a control. * indicates p < 0.05 by one-way ANOVA with Newman–Keuls post-hoc analysis (n 5 18 for all samples from three animals).

and the six mechanical parameters estimated from the r–E curves are shown in Figure 3(B). No significant differences in DE versus N samples were observed comparing the six

Seeding of pericardial patches with aortic valve-derived valve interstitial cells Various cell types have been proposed to repopulate decellularized tissue patches or biomaterials for heart valve engineering.41 This included endothelial cells, fibroblasts, and bone marrow-derived cells.26,42 Surprisingly, the cells normally deputed to valve leaflet tissue turnover and remodeling, the valve interstitial cells,43 have been only recently introduced in TEHV design.44 To assess feasibility of aVICs seeding into the DE pericardium, these cells were isolated from human and pig aortic valves. Before seeding, an immunophenotyping of the human cells was also performed by flow cytometry and immunofluorescence. These tests showed the expression of conventional aVICs markers Vimentin and aSMA [Figure 5(A)], and a minimal contamination by endothelial cells, as assessed by a low level of CD31 expression [Figure 5(B)]. Interestingly, the cells expressed high levels of CD44, CD73, CD90, and CD105 mesenchymal markers45 and, consistently with other reports,46 they only showed the class I (HLA-ABC), but not the class II (HLA-DR) MHC antigens. Pig-derived valve cells were not characterized in parallel due to the lack of suitable antibody cocktails.

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FIGURE 3. (A) Representative r–E curves for native (N), decellularized (DE) and Glutaraldehyde-fixed (GA) porcine pericardium tissue samples obtained from UTL tests. (B) The six parameters extracted for tissue biomechanical characterization are highlighted in red: elastic modulus at low (Elow) and high (Ehigh) strain values, calculated as the slope of the linear regression lines (red solid lines) of the two linear regions of the r–E curves, transition strain (Etrans, corresponding to the intersection point of the linear regression line with the abscissa) and stress (rtrans, that is, the r value corresponding to Etrans), and maximum tensile stress (rmax) and strain (Emax). (C) Results are graphically represented by average and standard errors. * and # indicate a p < 0.05 significance level in the comparison between DE and/or GA versus N and GA versus DE and N samples, respectively, by one-way ANOVA comparisons with Newman–Keuls post hoc analysis (n 5 18 for all samples from three animals).

The parietal pericardium has two surfaces, the serosa (S) and the fibrosa (F), which are exposed, respectively, to the pericardial cavity or the mediastinum. To assess possible

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differences in the efficiency of cell attachment, cells were seeded onto both surfaces of DE tissue patches at low and high concentrations and cultured for different times (24 and

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FIGURE 4. (A) Representative P–V curves for native (N), decellularized (DE) and glutaraldehyde-fixed (GA) porcine pericardium tissue samples acquired at 10 lL min21. (B) The four parameters extracted from P–V curve characterizing the tissue distensibility properties are highlighted in red: the P–V curve slope at low (P’low) and high (P’high), calculated as the slope of the linear regression lines (red solid lines) of the linear curve regions at low and high infused volumes, and the volume (Vtrans) and pressure (Ptrans) values at the transition between the two characteristic P– V curve slopes. Vtrans corresponds to the intersection point of the high slope linear regression line with the abscissa and Ptrans is the p values corresponding to Vtrans. (C) P–V curves acquired on DE samples at three different Q values: the plateau of the P–V curve (Pplateau) used for fluid tissue filtration (/) calculation is indicated in the red box. (D) Results are graphically represented by average and standard errors. * and # indicate a p < 0.05 significance level in the comparison between DE and/or GA versus N and GA versus DE and N samples, respectively, by one-way ANOVA comparisons with Newman–Keuls post-hoc analysis (n > 10 for all samples from three animals).

72 h) after mounting the pericardial patches into sterilized PDMS cartridges. The MTT metabolic staining revealed a higher ability of the cells to adhere to the S surface, when seeded with both cellular concentrations, especially at 72 h culture time [Figure 6(A,B)]. To observe the microscopic assessment of cell colonization through the cross-section, the cell-seeded patches on the S surface were fixed and embedded for histological sectioning. Nuclear and histological staining confirmed cells adherence to the S surface at the considered time points [Figure 6(C,D)], but not an indepth penetration into the tissue. For this reason, also con-

sidering the relatively short time of culture, we did not investigate the extracellular matrix deposition on the surface of the decellularized pericardium.

DISCUSSION

The manufacture of implants with a life-long duration and a maximized biological compatibility is still an unreached standard in the current scenario of heart valve bioprostheses. Despite the introduction of aldehyde-treated porcine valves and bovine/porcine pericardium has

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ameliorated the outcome of valve surgery, especially in elderly patients,3 there is still an active search of more suitable materials. These may be used to produce implants, for example, for young patients with congenital valve malformation, who have stronger immune reactions against the biological implants, and show a higher rate of structural valve deterioration.5,15,47

FIGURE 5. aVICs characterization by immunofluorescence and flow cytometry. (A) Presence of polymerized aSMA (green fluorescence) and Vimentin intermediate filaments (red fluorescence), as detected by confocal microscope in human valve-derived aVICs; blue fluorescence: nuclear staining (DAPI). (B) Flow cytometry analysis of human aVICs. The grey histograms show the fluorescence profile of cells stained with the indicated antibodies and are overlapped to those resulting from staining with the Isotype control antibodies (black histograms). The percentage of cells expressing HLA-ABC, HLA-DR, CD31, CD44, CD73, CD90, and CD105 are indicated above each histogram (average 6 standard error; n 5 3).

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Fixative free decellularization procedure abolishes aGAL content and ensures the maintenance of mechanical properties of pig-derived pericardium The key aspects in chronic rejection of aldehyde-fixed tissues employed in the manufacture of valve bio-prostheses are the presence of fixative residues in the porcine biological valves or bovine and porcine pericardial tissue,48 as well as of xenoantigens that are not removed by the fixation procedure.49 The adoption of refined strategies such as the employment of low-dose glutaraldehyde,11 short aldehyde treatment times,50 or treatment with detergents, protein solubilization procedures or metal coating12,13,51–54 has allowed to circumvent the problems inherent to GA toxicity, but have not resolved those inherent to the presence of xenoantigens. For its proof of principle design, the present research did not address the overall content of cell- and matrix-associated immunogens, or of matrix-related valve disease markers. To do this, a more accurate determination of, for example, the glycosphyngolipids55 and/or proteoglicans/GAGs56 content in the decellularized tissue would have to be performed by proteomic methods. Moreover, the impact of these components on cell-mediated immune reaction would have to be carefully evaluated by in vitro and in vivo testing before considering immunologically safe the decellularized tissue for a perspective in human transplantation. On the other hand, the evidence (Figure 2) that the decellularization procedure adopted for the porcine pericardium reduced below the detection limit the content of the 1,3 a-galactose,6–9 confirms the potential advantage of osmotic/detergent decellularization procedures to strongly reduce the level of xenoantigens and enhance immunological tolerance.10,21 Consistently with the report where our decellularization strategy was first described,21 the procedure adopted in the present investigation maintained the tissue native mechanical properties, as assessed by UTL and distensibility tests (Figures 3 and 4). Of note, the P–V curve analyses revealed significant differences in the permeability properties of DE pericardium that may have implications for the manufacture of cellularized bioprosthetic valves. In fact, the increase in tissue permeability might be exploited to favor an in-depth cell colonization of the cells by applying a suitable mechanical stimulus (for example, controlled medium circulation) allowing cells initially seeded on the surface to colonize the inner portion of the pericardium and favor the deposit of valve-specific extracellular matrix components. In this way, the conditions for chronic rejection may be reduced, as reported for the pericardium of a-Gal knockout pigs,36 while enabling to dynamically seed cells able to colonize the tissue in its entire depth.

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FIGURE 6. MTT staining of the porcine aVICs-seeded pericardial samples using low (0.6 3 103 cell mm22) and high (3 3 103 cell mm22) cellular density at the indicated time points. (A) represents the result of cell seeding on the fibrosa (F) surface while (B) shows the outcome of aVICs seeding on the serosa (S) side. The difference in the level of MTT staining of the seeded patches, observed especially at 72 h, suggests a higher adhesion of the cells to the S surface. (C) Histological sectioning of DE pericardium seeded with the high pig aVICs concentration at 1 and 3 days post-seeding. The images show the nuclear staining of the cells (arrows) overlapped with phase contrast pictures at lower (left) and higher (right) magnification. S and F indicate the serosa and the fibrosa surfaces, respectively. (D) Histological sectioning of the pericardium seeded with human aVICs at 3 days post-seeding. The upper image shows the DAPI/phase contrast image of the cell-seeded pericardium while the lower image shows a trichrome Masson’s staining of an adjacent section. S and F indicate the serosa and the fibrosa surfaces, respectively.

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Feasibility of aVICs seeding onto decellularized pericardium: A novel way to obtain TEHVs with cells naturally deputed to valve homeostasis Apart from improvements in the preparation procedures of the currently employed materials for valve implant manufacture, a next step in bioprosthetic valve design is expected from the generation of living valve leaflet tissues that may be used in spite of the fixed pericardium. The inclusion of cells in valve bio-prostheses manufacturing process may be in fact crucial to ensure the ability to self-renew and selfrepair typical of the natural leaflets. Recent examples of “off-the-shelf” TEHVs manufactured by combining bioartificial materials and cells with progenitor characteristics have been provided and preclinically tested.57,58 This latter approach, however, has to take into account the need for a rigorous biomaterial testing form a mechanical point of view and an extensive biological validation of the bioartificially reconstituted leaflet tissue before clinical transfer. An attractive alternative to the employment of complex biomaterial–cell formulations is, therefore, the devise of recellularization strategies of tissues that are already commercially employed for the manufacture of bioprosthetic valves. In this way, the use of (stem) cells would be restricted to seeding into tissues already validated for valve mechanical compliance, while the implants fabrication would be readily adapted from existing valve manufacture systems. The choice of the correct cell type to be employed in a recellularization strategy appears also relevant. Surprisingly, with few and recent exceptions,44,59,60 cell types that have been proposed and employed to this purpose41 are non-resident valve cells. Our choice to investigate the feasibility of aVICs seeding onto decellularized pericardium goes in the direction of finding suitable conditions to obtain a controlled colonization of the pericardial tissue by cells that maximally comply with the valve physiology. Because these cells possess the predisposition to valve mechanosensing61–63 and synthesize natural valve extracellular matrix components such as collagen and proteoglycans they may represent the best cell type minimizing the risks of abnormal calcification at mid- and long-term due to osteogenic differentiation. An additional advantage may also derive from a relatively high compatibility of these cells compared to other cell types for allogenic transplantation. In fact, unlike endothelial cells, and similarly to bone marrowderived mesenchymal progenitors,64 these cells do not express class II MHC [Figure 5(A)] and do not elicit cellmediated immune response, even under inflammatory stimulation.46 This provides the hope that employment of GMPamplified and banked allogenic human aVICs for seeding decellularized animal pericardium patches, might represent a realistic option to manufacture an immunologically tolerated valve tissue. The advantage of pig-derived aVICs may finally consist in the option to employ, as donors, genetically modified animals lacking xenoantigen expression.36 In this way, cells from genetically modified animals may be employed for large scale production of bioprosthetic implants. Future studies are planned by our groups in order to address these possible alternatives.

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Although the results presented in our study only show a short-term aVICs culture on the surface of both surfaces of the pericardium without a long-term analysis of the cell penetration and matrix deposition, they demonstrate the potential of these cells to adhere to fixative-free decellularized pericardium, and in particular to its serosa surface (Figure 6). In addition, the relatively high tissue permeability observed in the decellularized pericardium (Figure 4), raises the possibility that application of a forced culture medium circulation may be exploited to deliver valve-competent cells inside the pericardial membrane. In preparation of these future developments, we have adopted a standardized VICs seeding procedure directly into bioreactor posts which may be easily transferred to dynamic culturing. All these evidences represent a novelty compared with previous approaches and establish novel criteria for future bioreactor-assisted dynamic cell seeding into the pericardium, and to obtain a leaflet-like maturation of the engineered tissue for TEHVs manufacture. REFERENCES 1. Binet JP, Carpentier A, Langlois J. Clinical use of heterografts for replacement of the aortic valve. J Thorac Cardiovasc Surg 1968; 55:238–242. 2. Carpentier A, Blondeau P, Laurens B, Hay A, Laurent D, Dubost C. Mitral and tricuspid valve replacement with frame-mounted aortic heterografts. J Thorac Cardiovasc Surg 1968;56:388–394. 3. Forcillo J, Pellerin M, Perrault LP, Cartier R, Bouchard D, Demers P, Carrier M. Carpentier-Edwards pericardial valve in the aortic position: 25-years experience. Ann Thorac Surg 2013;96:486–493. 4. Grabenwoger M, Sider J, Fitzal F, Zelenka C, Windberger U, Grimm M, Moritz A, Bock P, Wolner E. Impact of glutaraldehyde on calcification of pericardial bioprosthetic heart valve material. Ann Thorac Surg 1996;62:772–777. 5. Siddiqui RF, Abraham JR, Butany J. Bioprosthetic heart valves: Modes of failure. Histopathology 2009;55:135–144. 6. Konakci ZK, Bohle B, Blumer R, Hoetzenecker W, Roth G, Moser B, Boltz-Nitulescu G, Gorlitzer M, Klepetko W, Wolner E, et al. Alpha-Gal on bioprostheses: Xenograft immune response in cardiac surgery. Eur J Clin Investig 2005;35:17–23. 7. Konakci KZ, Bohle B, Blumer R, Hoetzenecker W, Roth G, Moser B, Boltz-Nitulescu G, Gorlitzer M, Klepetko W, Wolner E, Ankersmit HJ. First quantification of alpha-Gal epitope in current glutaraldehyde-fixed heart valve bioprostheses. Xenotransplantation 2013;20:252–261. 8. Naso F, Gandaglia A, Iop L, Spina M, Gerosa G. Alpha-Gal detectors in xenotransplantation research: A word of caution. Xenotransplantation 2012;19:215–220. 9. Galili U. The [alpha]-gal epitope and the anti-Gal antibody in xenotransplantation and in cancer immunotherapy. Immunol Cell Biol 2005;83:674–686. 10. H€ ulsmann J, Gr€ un K, El Amouri S, Barth M, Hornung K, Holzfuß C, Lichtenberg A, Akhyari P. Transplantation material bovine pericardium: Biomechanical and immunogenic characteristics after decellularization vs. glutaraldehyde-fixing. Xenotransplantation 2012;19:286–297. 11. Sinha P, Zurakowski D, Susheel Kumar TK, He D, Rossi C, Jonas RA. Effects of glutaraldehyde concentration, pretreatment time, and type of tissue (porcine versus bovine) on postimplantation calcification. J Thoracic Cardiovasc Surg 2012;143:224–227. 12. Guldner NW, Bastian F, Weigel G, Zimmermann H, Maleika M, Scharfschwerdt M, Rohde D, Sievers HH. Detoxification and endothelialization of glutaraldehyde-fixed bovine pericardium with titanium coating. Circulation 2009;119:1653–1660. 13. Lim H-G, Kim SH, Choi SY, Kim YJ. Anticalcification effects of decellularization, solvent, and detoxification treatment for genipin

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