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Ann Thorac Surg 2003;75:1274-1282
© 2003 The Society of Thoracic Surgeons

Original article: cardiovascular

Cell characterization of porcine aortic valve and decellularized leaflets repopulated with aortic valve interstitial cells: the VESALIO project (Vitalitate Exornatum Succedaneum Aorticum Labore Ingenioso Obtenibitur)

Barbara Bertipaglia, PhDa, Fulvia Ortolani, PhDb, Lucia Petrelli, ScDb, Gino Gerosa, MDc, Michele Spina, MDd, Paolo Pauletto, MDa, Dino Casarotto, MDc, Maurizio Marchini, PhDb, Saverio Sartore, PhDd*

a Department of Experimental and Clinical Medicine, Padua, Italy
d Department of Biomedical Sciences, Padua, Italy
c Institute of Cardiovascular Surgery, University of Padua, Padua, Italy
b Department of Medical and Morphological Research, University of Udine, Udine, Italy

Accepted for publication October 24, 2002.

* Address reprint requests to Dr Sartore, Department of Biomedical Sciences, University of Padua, Viale G. Colombo 3, I-35121 Padua, Italy.
e-mail: sartore{at}civ.bio.unipd.it


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: Heart valve bioprostheses for cardiac valve replacement are fabricated by xeno- or allograft tissues. Decellularization techniques and tissue engineering technologies applied to these tissues might contribute to the reduction in risk of calcification and immune response. Surprisingly, there are few data on the cell phenotypes obtained after cellularizing these naturally-derived biomaterials in comparison to those expressed in the intact valve.

METHODS: Aortic valve interstitial cells (VIC) were used to repopulate the corresponding valve leaflets after a novel decellularization procedure based on the use of ionic and nonionic detergents. VIC from leaflet microexplants at the third passage were utilized to repopulate the decellularized leaflets. Intact, decellularized and repopulated valve leaflets and cultured VIC were examined by immunocytochemical procedures with a panel of antibodies to smooth muscle and nonmuscle differentiation antigens. Intact and cellularized leaflets were also investigated with Western blotting and transmission electron microscopy, respectively.

RESULTS: Myofibroblasts and smooth muscle cells (SMC) were mostly localized to the ventricularis of the leaflet whereas fibroblasts were dispersed unevenly. Cultured VIC were comprised of myofibroblasts and fibroblasts with no evidence of endothelial cells and SMC. Two weeks after VIC seeding into decellularized leaflets, grafted cells were found penetrating the bioscaffold. The immunophenotypic and ultrastructural properties of the grafted cells indicated that a VIC heterogeneous mesenchymal cell population was present: fibroblasts, myofibroblasts, SMC, and endothelial cells.

CONCLUSIONS: VIC seeding on detergent-treated valve bioscaffolds has the cellular potential to reconstruct a viable aortic valve.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 Acknowledgments
 References
 
Tissue-engineered bioprosthesis prepared from natural healthy tissues in comparison with the mechanical valve substitutes, xenogenic bioprostethic, and cryopreserved allograft valves may have some advantages, such as a reduction in thromboembolism, inflammatory response, and calcification [1, 2].

Preservation of the original tissue make-up can be accomplished by treating the valve to be transplanted with either a cross-linking agent or dye-mediated photooxidation [2]. Antigenicity of these transplants can be controlled to some extent with decellularization of the original valve by enzymatic or detergent methods [3, 4],possibly in combination with a chemical fixation to reduce calcification [2]. This procedure provides a bioscaffold that can be seeded with the appropriate cell population(s) to reconstitute the original valve [4, 5]. An alternative tissue-engineered strategy to fabricate a viable and functional valve relies on a chemically-defined biodegradable scaffold that is cellularized using autologous cells [6–10]. Both techniques assume that transplanted cell populations in the bioscaffolds/biosynthetic scaffolds achieve the same (or very similar) topographic distribution and differentiation pattern of the naturally-occurring VIC in the original valve [11].

Surprisingly, only a few studies have dealt with such issues. VIC have been identified as myofibroblasts (cell phenotype having some properties in common with fibroblasts and SMC) [11–16] or SMC [16, 17] with a poor definition of spatial distribution among the three valve tissues, ventricularis, spongiosa and fibrosa. There is almost no indication as to which myofibroblast or SMC variant populates the valve tissues [18, 19]. For seeding, some authors have used "vascular cells" [10, 20], fibroblasts [21], fibroblasts+endothelial cells (EC) [6, 8], fibroblasts followed by EC [5, 9, 11], and myofibroblasts+EC [5, 7]. In these studies there is almost no data regarding the phenotypic profile once these cells are transplanted into the scaffolds [5].

We previously identified the topographic distribution of VIC in human semilunar valves [16]. As a first step in using selected cell populations to seed bioscaffolds, we report on the phenotypic characterization and localization of VIC in normal porcine valves and the ability of cultured allo-VIC to repopulate a decellularized valve matrix in vitro. Decellularization of aortic valve leaflets (AVL) is achieved with a new procedure that uses Triton X-100 and sodium cholate [22]. The phenotypic profile of VIC to be used in the transplantation experiments has been studied before (in vitro) and after (in vivo) grafting these cells to the AVL. Our results indicate that VIC can successfully repopulate the detergent-treated matrices and can give rise to a heterogeneous mesenchymal cell population in the allograft: fibroblasts, myofibroblasts, SMC, and EC.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 Acknowledgments
 References
 
Collection of samples
Aortic valves and AVLs from adult pigs (n = 7; mean age and weight were 18 weeks and 80 kg, respectively) were used for in vitro studies and in cell repopulation protocols of decellularized valve matrices.

Electrophoresis and Western blotting
Single AVL from adult pigs (n = 5) were finely minced with forceps before being frozen in liquid nitrogen. Frozen fragments were powdered in a mortar and subsequently mixed with Laemmli’s sample solution before finally being boiled to allow solubilization of AVL components. Aortic smooth muscle (SM) tissue was also extracted for comparison. Electrophoresis was performed in 5% or 12.5% SDS gels and Western blotting was carried out as previously described, in quadruplicate [23], using the panel of antibodies described below. Nonimmune IgG were used as the control.

Immunocytochemistry
AVL cryosections (9-µm thick) were collected on precoated gelatin slides. A panel of monoclonal/polyclonal antibodies, cross-reacting with the porcine tissues, were selected because of their ability to identify the major cell types present in human semilunar valves [16]. The following antibodies were used: SM-E7 anti-SM myosin heavy chains (MyHC; specific for SM1 and SM2 isoforms, respectively) [19, 24]; NMF6 antiplatelet type MyHC-Apla1 [19, 24]; NM-G2 antiplatelet type MyHC-Apla2 [19, 24]; and E11 antiSM22 [23]. The antivimentin (clone V9) and anti-SM {alpha}-actin (clone 1A4) were purchased from Sigma (St. Louis, MO), the antivon Willebrand factor (anti-vWf) from Dako (Dakopatts, Glostrup, Denmark) and the anti-thrombomodulin (anti-TM) from Serotec Ltd. (Oxford, UK). We followed the procedure described in [16, 23]. Bound antigens were revealed by 3-amino-9-ethylcarbazole (Sigma).

Decellularization protocol
Detergent-based decellularization procedure was carried out as previously described [22] with minor modifications to be reported elsewhere (submitted). Phosphate–Buffered Saline (PBS)-washed AVLs were extracted in the presence of protease inhibitors in degassed solutions containing Triton X-100 and sodium cholate in a nitrogen atmosphere. After detergent treatment in some valve preparations (n = 3), nucleic acids were removed by Benzonase (Merck, Darmstadt, Germany), a recombinant endonuclease that degrades single- and double-stranded DNA and RNA.

Repopulation protocol
Tissue culture
Both left and right cusps of aortic valves were used in this experiment. Endocardial (EC) were first mechanically removed from AVLs by a cotton rubber and then by treating AVLs with 0.2% collagenase A (Roche Diagnostics, Indianapolis, IN) in Dulbecco’s Modified Eagle’s Medium (DMEM) for 10 minutes at 37°C. Removal of EC is confirmed by immunofluorescence assay using "en-face" preparation of AVLs reacted with anti-vWf antibody (not shown). De-endothelialized AVLs were minced into 1- to 2-mm pieces and evenly distributed in tissue culture dishes (Falcon) covered with fibronectin (50 µg/ml, Roche) in PBS, pH 7.2. The cell culture medium consisted of 10% fetal calf serum (FCS; Sigma), 5% of penicillin-streptomycin solution, 5% of L-glutamine solution, and 5% of Hepes solution in DMEM. Cells migrated off the explants (after 1 week) were grown up to a preconfluent state and then serially passaged to obtain enough cells for cell seeding experiments (the third passaged cells were used on AVL decellularized matrices and identified as VIC).

Individual decellularized AVL were placed into 24-well multiwell plates, rinsed with PBS and then incubated for 12 hours with DMEM and for another 12 hours with FCS. After this step, each matrix was pretreated with fibronectin (50 µg/ml) for 24 hours. Eight decellularized AVLs per experiment (five distinct repopulation experiments) were seeded in multi-well plates with 2x106 VIC/AVL using short pulses of mild shaking during the first 24 hours and leaving the AVLs in static conditions thereafter. The culture media was the same as described above but, in addition, contained the Site Liquid Media Supplement (1x, Sigma). After 15 days of incubation the seeded valves were washed in PBS and then fixed with 4% p-formaldehyde for histologic, immunocytochemistry, and electronic transmission microscopy studies.

Histologic analysis
Strips about 3-mm wide were excised from repopulated AVL along their longitudinal axis, de-hydrated in graded ethanols and embedded in paraffin. 5-µm thick sections were stained with hematoxylin-eosin stain and Movat’s stain.

Ultrastructural analysis
Both decellularized and decellularized+repopulated AVL samples were examined ultrastructurally. Specimens of about 1 mm2 x 0.5 mm were fixed with 2.5% formaldehyde-2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.2, postfixed with 2% OsO4 in the same buffer, dehydrated with graded ethanols and embedded in Araldite/Epon. Thin sections were contrasted with uranyl acetate and lead citrate. Observations and photographic records were made with a Philips CM12 STEM electron microscope (Philips Export BV, Eindhoven, Holland).

Immunofluorescence analysis
Before being used for cell seeding of decellularized AVLs, VIC obtained from AVL explants were evaluated for the immunophenotypic profile using the antibodies described above. Primary or third passaged VIC were fixed with 2% p-formaldehyde in PBS, pH 7.4 for 10 minutes and permeabilized with Triton X-100 (Sigma) for a few seconds [23]. Primary antibodies were used as previously reported [23]. After rinsing with PBS, the slides were treated with an antimouse or antirabbit IgG coupled with tetramethylrhodamine isothiocyanate (Dako, Hamburg, Germany), under the condition described above. Slides were rinsed in PBS and then mounted in elvanol and examined by a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Inc., Thornwood, NY).


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 Acknowledgments
 References
 
Distribution of differentiation markers in the intact AVL
SDS extracts from normal specimens of AVL were first studied in Western blotting (Fig. 1) using a panel of monoclonal antibodies to some SM and platelettype nonmuscle (NM) differentiation markers [18, 19, 23]. SM {alpha}-actin and vimentin immunostaining are clearly detectable in the Western blots (Fig. 1A), whereas SM22 is less detectable. In 5% gels, MyHC-Apla1 and, to a lesser extent, MyHC-Apla2 are seen, whereas SM myosin immunoreactivity is lacking. Based on accepted cell nomenclature [18, 19] these data are compatible with the presence of myofibroblasts in normal AVL (see also Table 1).



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  		Fig 1. Electrophoresis and Western blotting (a, b, and c) of aortic valve leaflet (AVL) and aortic smooth muscle (SM) tissue extracts examined with a panel of antibodies. (A) 12.5% sodium dodecyl sulfate (SDS) gel. (B) 5% SDS gel. Lane 1, aortic extract; lane 2, AVL extract. Note in AVL extracts the actin, vimentin, and SM22 band (A), the absence of SM myosin band and the presence of platelet myosin isoforms with different intensity (B). (MyHC-Apla1 = type-1 platelet myosin heavy chains; MyHC-Apla2 = type-2 platelet myosin heavy chains.)

 

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  		Table 1. VIC Phenotype Composition in Intact and Cell Repopulated Aortic Leaflets in Comparison With VIC Grown In Vitro

 
The phenotypic profile and the spatial distribution of cells in the left coronary cusp from normal AVL are illustrated in Figure 2. Although all cells in the three-tissue valve (ventricularis, spongiosa, and fibrosa [not shown]) are labeled for vimentin (Fig. 2A) the two NM myosins (Fig. 2C and 2D), SM {alpha}-actin (Fig. 2B) and SM22 (Fig. 2E) can be mostly found in clusters in the spongiosa. SM myosin is localized to the ventricularis in a tiny areas (Fig. 2F). Thus, different types of fibroblasts (vimentin+, vimentin+/MyHCA-pla1+/MyHCA-pla2+) along with myofibroblasts (vimentin+/SM {alpha}-actin+/SM22+) and a small population of SMC (vimentin+, SM {alpha}-actin+, SM22+, and SM myosin+) [18, 19, 23, 24] are segregated in a tissue-specific manner in the valve cusp.



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  		Fig 2. Immunocytochemical staining of aortic valve leaflet from the left coronary cusp (middle part, ventricularis and spongiosa) reacted with a panel of antibodies (A–I). Valve interstitial cells are all positive for vimentin, MyHC-Apla1, and -Apla2. Differences in the distribution of labeled cells among the different cusps can be seen for smooth muscle (SM) {alpha}-actin and SM22. Arrows in F indicate SM myosin+ cells. Note that myofibroblasts (*) and SM cells are localized to the outermost layer of the cusp. Bar = 150 µm. (e = endothelium; MyHC-Apla1 = type-1 platelet myosin heavy chains; MyHC-Apla2 = type-2 platelet myosin heavy chains; niIgG = non-immune IgG; TM = thrombomodulin; vWf = von Willebrand factor.)

 
Immunophenotypic characterization of cultured VIC
Valve interstitial cells from AVL grown in vitro to be used in repopulation experiments exhibit a mixture of cell phenotypes: spindle-shaped cells (Figs 3A–3C, and 3E) or clusters of cobblestone-like cells (Fig 3G) that resemble the so-called epithelioid cells described for cultured SMC [19]. The cultured VIC display a markedly lower reactivity for MyHC-Apla2 (Fig 3D) and most of them are positive for SM22 (Fig 3E). In spindle-shaped and cobblestone-like cells, there is neither SM myosin nor vWf (Fig 3H) or TM (not shown) expression. Altogether these data indicate that fibroblasts and myofibroblasts are the predominant cell phenotype expressed in the cultured VIC (Table 1). This pattern is clearly distinguishable from the one of cultured SMC (composed of myofibroblasts and immature SMC) [19, 23] and adventitial fibroblasts (composed of fibroblasts and myofibroblasts with a poor expression of SM22 [23]).



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  		Fig 3. Immunofluorescence staining (A–F, H) of valve interstitial cell cultures from aortic valve leaflet explants obtained at the third passage. Note that although most cells are vimentin, MyHC-Apla1, and SM22 positive, none express smooth muscle (SM) myosin or von Willebrand factor (vWf). A proportion of cells are stained for actin and only very weakly for MyHC-Apla2. (G) Phase contrast of panel H. The red line encircles a typical cluster of epithelioid cells. Bars in A–F = 50 µm; bars in G–H = 30 µm. (MyHC-Apla1 = type-1 platelet myosin heavy chains; MyHC-Apla2 = type-2 platelet myosin heavy chains.)

 
Structural features of decellularized matrices
Histologic analysis demonstrates that intact VIC nuclei are undetectable in detergent-treated bioscaffolds after benzonase treatment (Fig. 4C). However, a minor contamination with nucleic acid aggregates can be visualized by immunofluorescence staining with Hoechst 33258-trihydrochloride (not shown). In comparison with the intact AVL (Fig. 4B), the decellularized matrices (Fig. 4D) reveal a good histochemical preservation with Movat staining that is also confirmed ultrastructurally through examination of the major components of extracellular matrix (Fig. 5).



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  		Fig 4. Hematoxylin-eosin (H&E) staining (A, C, E) and Movat staining (B, D, F) of intact (A, B), decellularized (C, D), and decellularized+repopulated (E, F) aortic valve leaflets (AVL). Decellularization procedure was carried out in the presence of benzonase. Note that Movat stains the elastic fibers purple, the collagen fibers pink, and the proteoglycans blue. Bar = 100 µm. Box a in panel (E) indicates the valve region used for ultrastructural studies shown in Fig 7.

 


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  		Fig 7. Ultrathin sections of decellularized/repopulated aortic valve leaflet. (A) Two endothelial-like cells interacting with the bioscaffold join together (arrow) overpassing a furrow lined by basal lamina (arrowheads). (B) Endothelial-like cells closely adhering to basal lamina-like layer (double arrows). (C) A cell junction (arrow) is developing between two endothelial-like cells (double arrows). (D) Cell junctions between two myofibroblasts, or smooth muscle-like cells (arrowheads). (E) Oblong myofibroblast. (F) Myofibroblast demonstrating abundant microfilaments. (G –I) Fibroblast-like cells; note fibrillin microfibrils surrounding developing elastin fibers (double arrowheads) and collagen-fibril-forming channels (arrowheads). Bars: A, B, D, H, and I = 1 µm; C and F = 0.5 µm; E and G = 3 µm.

 


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  		Fig 5. Electron micrograph illustrating a decellularized aortic valve leaflet treated with benzonase revealing normal appearing collagen fibrils (c), elastic fibers (e), and fibrillin microfibrils (f). Bar = 0.5 µm.

 
Phenotypical features of decellularized and repopulated matrices
Grafted cells are able to adhere and actively migrate, though to a variable extent, into the decellularized matrix (Fig. 4E) and differentiate in a spatial-specific manner as demonstrated by immunocytochemical (Fig 6) and ultrastructural (Fig 7) results.



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  		Fig 6. (A–I) Immunocytochemical identification of cells repopulating aortic valve leaflet bioscaffolds from left coronary cusp (middle part, ventricularis and spongiosa; images were taken from a region similar to that illustrated in Fig 4E, square "a"). Note that the majority of cells are stained for vimentin whereas fewer cells are actin+, rare are smooth muscle (SM) myosin+ (arrow) and none are stained for the two anti-MyHC-Apla isoforms. Anti-SM22, anti-von Willebrand factor (anti-vWf) and anti-TM antibody labeled a thin rim of transplanted cells. The latter also stains inner cells (asterisk). Bar = 110 µm. (MyHC-Apla1 = type-1 platelet myosin heavy chains; MyHC-Apla2 = type-2 platelet myosin heavy chains; niIgG = non-immune IgG; TM = thrombomodulin.)

 
Cells penetrating AVL bioscaffolds are positive for vimentin, SM {alpha}-actin, and to a lesser extent, SM22. SM myosin is expressed in few traceable amounts (Fig 6F). MyHC-Apla1 and -Apla2 are negative with the transplanted cells. Interestingly, vWf (Fig 6G) and TM (Fig 6H) are mostly expressed at the level of the AVL surface along with SM22, though a TM immunostaining was also observed in the inner part of the repopulated AVL. Thus, a multilayered distribution of VIC immunophenotypes can be seen in the repopulated AVL bioscaffolds: a mixed endothelial/SM-like cell (outermost layer) and a myofibroblast/fibroblast cell (innermost layer) phenotype (Table 1).

Ultrastructurally, endothelial-like cells were observed to lie near basal lamina-like membranes (Figs 7A and 7B) and cell junction development is apparent in some contact areas (Fig 7C). Within the AVL, the moving cells characterize a branched cytoplasm with pseudopodelike protrusions, whereas other cells display either fibroblastic-like (Fig 7G) or spindle-like (Fig 7E) features. Involvement of the fibroblasts in extracellular matrix remodeling is documented by the presence of a dilated rough endoplasmic reticulum as well as a number of secretion vesicles opening at cell surface (Fig 7H). Numerous collagen fibrils containing intracellular channels, which resemble those in the sites of collagen fibrillogenesis, are observed (Fig 7I). Moreover, elastogenesis is also in progress as shown by irregular fibers of amorphous elastin intermingled with a notable number of fibrillin microfibrils (Figs 7B and 7H). As for spindle-shaped cells, accumulation of cooriented bundles of actin-like microfilaments is indicative of myofibroblast (Fig 7F). Development of cell junctions in these cells suggests a potential differentiation to SMC (Fig 7D).


   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
Acknowledgments
 References
 
For a decellularized valve matrix to be suitable as in vivo substitute it must be viable and mechanically performing [25]. Moreover, this matrix should allow for engrafting, proliferation, differentiation, surviving and spatial distribution of exogenous seeded cells to reproduce the morphofunctional organization of the naturally occurring valve. Minimally modified AVL bioscaffolds prepared by our detergent treatment leave a great deal of the extracellular matrix components in situ also creating local structural conditions for active cell homing. This is confirmed by the appearance of intracytoplasmic collagen-fibril-forming channels and elastogenesis occurrence (Fig 7) in repopulated matrices. Preliminary in vivo function studies indicate that these bioscaffolds preserve biomechanical properties and scarce inflammatory response (in preparation).

We have also demonstrated that decellularized AVL matrices prepared by detergent treatment can be actively colonized by cultured VIC giving rise to four distinct mesenchymal cell populations: fibroblasts, myofibroblasts, SMC, and EC. These phenotypes indeed correspond to the original cellular components of the intact AVL in which myofibroblasts (and SMC) are mostly expressed at the level of ventricularis whereas the fibroblasts are localized unevenly throughout the spongiosa and fibrosa. EC, identified by vWf and TM expression, are correctly localized to the cellular lining covering the cellularized matrix. The limited time of static VIC culturing of decellularized AVL does not allow any firm conclusion about the propensity of fibroblasts, myofibroblasts and SMC to be spatially organized according to the original distribution exemplified in Figure 2. However, myofibroblasts and SMC appear to be localized, as in the intact AVL, in the outermost layers of transplanted VIC and the fibroblasts, correctly, seem to be part of the "leading edge" of migrating cells in the innermost region of the matrix. Interestingly, vWf and TM expression, as well as ultrastructural features of EC (Fig 7), could be evidenced only when VIC grow on decellularized matrix. This finding suggests that VIC possess a heterogeneous differentiation potential [26], which might be endowed by spindle-shaped and cobblestonelike cells present in vitro (Fig 3) [25]. Development of focal adhesions, extracellular fibronectin fibrils, and a cytocontractile apparatus in VIC as well as permissive structural conditions achieved with the detergent treatment of valve leaflets can confer these cells the ability to migrate deeply into the bioscaffold [27]. Propensity to migrate and colonize the bioscaffold could also be attributable to the specific microenvironment in which VIC were grown before being seeded onto the leaflets. The selected VIC subpopulation may in this circumstance undergo a phenotypic transition such as the epithelial-to-mesenchymal cell transdifferentiation observed in valvulogenesis. Both aortic embryonic endothelial [28] and endocardial [29] cells undergo a phenotypic conversion to SM {alpha}-actin expressing mesenchymal cells: the myofibroblasts. As this process is regulated by TGF-ß isoforms [30] these cytokines are also likely to be involved in the conversion to SMC [19], which may represent the final differentiation step of some myofibroblasts in the ventricularis tissue.

Our data are also compatible with the existence of a multipotent cell population, which is responsible for recreating the appropriate VIC and EC composition in the neovalve. Indeed, multiple cell types were used to colonize the scaffolds such as "vascular cells" [10, 20], fibroblasts [21], fibroblasts+ EC [6, 8], fibroblasts followed by EC [5, 9, 11], and myofibroblasts+EC [5, 7]. More recently, Taylor and coworkers [31] used human cardiac valve interstitial cells to seed collagen sponges. It seems plausible that the cell seeding procedure with the highest chances of establishing the cell repertoire and spatial distribution of intact valve should be the one that is close to the cell colonization pathway during valve morphogenesis. At the moment, two cell sources seem to be amenable for the repopulation procedure of xeno or allografts in humans. The adventitial fibroblasts and the bone marrow stem cells. Clearly the differentiation potential of these cells needs to be appropriately driven by predetermination treatments with growth factors, cytokines or specific differentiation inducers to channel their phenotypic potential towards the valve cells. With both procedures, however, it is possible for a further spatial-specific arrangement, once the whole valve is seeded, through a passage in a pulse duplicator. It is well known that mechanical forces including shear and tension stress, can modify the expression of SM {alpha}-actin and myosin isoforms and hence change the cell phenotypes [19, 27]. In this respect it is important to ascertain the stability of VIC phenotypes at prolonged times of culture. Thus, before surgical implantation of repopulated bioscaffolds, the cell differentiation profile must be determined in a spatial-specific manner with respect to the cell proliferation and surviving pattern in dynamic fluid conditions.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported by a special grant from Cassa di Risparmio di Padova e Rovigo Foundation, Padua, Italy and the Italian Space Agency, Rome, Italy. We wish to thank Abigail Johnson and Sarah Gacina for their excellent editorial assistance.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Schoen F.J. Future directions in tissue heart valves: impact of recent insights from biology and pathology. J Heart Valve Dis 1999;8:350-358.[Medline]
  2. Schmidt C.E., Baier J.M. Acellular vascular tissues: natural biomaterials for tissue repair and tissue engineering. Biomaterials 2000;21:2215-2231.[Medline]
  3. O’Brien M.F., Goldstein S., Walsh S., et al. The SynerGraft valve. A new a cellular (nonglutaraldehyde-fixed) tissue heart valve for autologous recellularization first experimental studies before clinical implantation. Sem Thorac Cardiovasc Surg 1999;11:194-200.[Medline]
  4. Stock U.A., Vacanti J.P. Tissue engineering: current state and prospects. Annu Rev Med 2001;52:443-451.[Medline]
  5. Steinhoff G., Stock U., Karim N., et al. Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduit. Circulation 2000;102(Suppl III):III-50-55.
  6. Shinoka T., Breuer C.K., Tanel R.E., et al. Tissue engineering heart valves: valve leaflet replacement study in a lamb model. Ann Thorac Surg 1995;60(Suppl 6):S513-516.
  7. Zund G., Breuer C.K., Sninoka T., et al. The in vitro construction of a tissue engineered bioprosthetic heart valve. Eur J Cardiothorac Surg 1997;11:493-497.[Abstract]
  8. Sninoka T., Shum-Tim D., Ma P.X., et al. Tissue-engineered heart valve leaflets. Does cell origin affect outcome?. Circulation 1997;96(Suppl II):II-102-107.
  9. Zund G., Hoerstrup S.P., Schoeberlein A., et al. Tissue engineering: a new approach in cardiovascular surgery: seeding of human fibroblasts followed by human EC on resorbable mesh. Eur J Cardiothorac Surg 1998;13:160-164.
  10. Sodian R., Hoerstrup S.P., Sperling J.S., et al. Early in vivo experience with tissue-engineered trileaflet heart valves. Circulation 2000;102(Suppl III):III-22-29.
  11. Hoerstrup S.P., Sodian R., Deabritz S., et al. Functional living trileaflet heart valves grown in vitro. Circulation 2000;102(Suppl III):III-44-49.
  12. Filip D.A., Radu A., Simionescu M. Interstitial cells of the heart valves possess characteristics similar to smooth muscle cells. Circ Res 1986;59:310-320.[Abstract/Free Full Text]
  13. Messier R.H., Jr, Bass B.L., Aly H.M., et al. Dual structural and functional phenotypes of the porcine aortic valve interstitial population: characteristics of the leaflet myofibroblasts. J Surg Res 1994;57:1-21.[Medline]
  14. Mulholland D.L., Gotlieb A.I. Cardiac valve interstitial cells: regulator of valve structure and function. Cardiovasc Pathol 1997;6:167-174.
  15. Roy A., Brand N.J., Yacoub M.H. Molecular characterization of interstitial cells isolated from human heart valves. J Heart Valve Dis 2000;9:459-464.[Medline]
  16. Della Rocca F., Sartore S., Guidolin D., et al. Cell composition of the human pulmonary valve: a comparative study with the aortic valve. The VESALIO project. Ann Thorac Surg 2000;70:1594-1600.[Abstract/Free Full Text]
  17. Bairati A, DeBiasi S. Presence of a smooth muscle system in aortic valve leaflets. Anat Embryol 1981;161:329–340
  18. Schmitt-Gräff A., Desmouliere A., Gabbiani G. Heterogeneity of myofibroblast phenotypic features: an example of fibroblastic cell plasticity. Virchows Arch 1994;425:3-24.[Medline]
  19. Sartore S., Franch R., Roelofs M., et al. Molecular and cellular phenotypes and their regulation in smooth muscle. Rev Physiol Biochem Pharmacol 1999;134:235-320.[Medline]
  20. Sodian R., Hoerstrup S.P., Sperling J.S., et al. Tissue engineering of heart valves: in vitro experiences. Ann Thorac Surg 2000;70:140-144.[Abstract/Free Full Text]
  21. Hoerstrup S.P., Zund G., Lachat M., et al. Tissue engineering: a new approach in cardiovascular surgery; seeding of human fibroblasts on resorbable mesh. Swiss Surg 1998;2(Suppl):23-25.
  22. Samouillan V., Dandurand-Lods J., Lamure A., et al. Thermal analysis characterization of aortic tissues for cardiac valve bioprostheses. J Biomed Mater Res 1999;46:531-538.[Medline]
  23. Faggin E., Puato M., Zardo L., et al. Smooth muscle-specific SM22 protein is expressed in the adventitial cells of the balloon-injured rabbit carotid artery. Arterioscler Thromb Vasc Biol 1999;19:1393-1404.[Abstract/Free Full Text]
  24. Sartore S., Chiavegato A., Franch R., et al. Myosin gene expression and cell phenotypes in vascular smooth muscle during development, in experimental models and in vascular disease. Arterioscler Thromb Vasc Biol 1997;17:1210-1215.[Abstract/Free Full Text]
  25. Durbin A.D., Gotlieb A.I. Advances towards understanding heart valve response to injury. Cardiovasc Pathol 2002;11:69-77.[Medline]
  26. Taylor P.M., Allen S.P., Yacoub M.H. Phenotypic and functional characterization of interstitial cells from human heart valves, pericardium and skin. J Heart Valve Dis 2000;9:150-158.[Medline]
  27. Tomasek J.J., Gabbiani G., Hinz B., et al. Myofibroblasts and mechanoregulation of connective tissue remodeling. Nature Rev Mol Cell Biol 2002;3:349-363.[Medline]
  28. DeRuiter M.C., Poelmann R.E., VanMunsteren J.C., et al. Embryonic endothelial cells transdifferentiate into mesenchymal cells expressing smooth muscle actins in vivo and in vitro. Circ Res 1997;80:444-451.[Abstract/Free Full Text]
  29. Eisenberg L.M., Markwald R.R. Molecular regulation of atrioventricular valvuloseptal morphogenesis. Circ Res 1995;77:1-6.[Free Full Text]
  30. Arciniegas E., Sutton A.B., Allen T.D., et al. Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro. J Cell Sci 1992;103:521-529.[Abstract]
  31. Taylor P.M., Allen S.P., Dreger S.A., Yacoub M.H. Human cardiac valve interstitial cells in collagen sponge: a biological three-dimensional matrix for tissue engineering. J Heart Valve Dis 2002;11:298-307.[Medline]



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