Human Mesenchymal Stem Cells Reendothelialize Porcine Heart Valve Scaffolds: Novel Perspectives in Heart Valve Tissue Engineering

Abstract Heart valve diseases are usually treated by surgical intervention addressed for the replacement of the damaged valve with a biosynthetic or mechanical prosthesis. Although this approach guarantees a good quality of life for patients, it is not free from drawbacks (structural deterioration, nonstructural dysfunction, and reintervention). To overcome these limitations, the heart valve tissue engineering (HVTE) is developing new strategies to synthesize novel types of valve substitutes, by identifying efficient sources of both ideal scaffolds and cells. In particular, a natural matrix, able to interact with cellular components, appears to be a suitable solution. On the other hand, the well-known Wharton's jelly mesenchymal stem cells (WJ-MSCs) plasticity, regenerative abilities, and their immunomodulatory capacities make them highly promising for HVTE applications. In the present study, we investigated the possibility to use porcine valve matrix to regenerate in vitro the valve endothelium by WJ-MSCs differentiated along the endothelial lineage, paralleled with human umbilical vein endothelial cells (HUVECs), used as positive control. Here, we were able to successfully decellularize porcine heart valves, which were then recellularized with both differentiated-WJ-MSCs and HUVECs. Data demonstrated that both cell types were able to reconstitute a cellular monolayer. Cells were able to positively interact with the natural matrix and demonstrated the surface expression of typical endothelial markers. Altogether, these data suggest that the interaction between a biological scaffold and WJ-MSCs allows the regeneration of a morphologically well-structured endothelium, opening new perspectives in the field of HVTE.


Introduction
In the past two decades, cardiac valve diseases have undergone deep changes, concerning the epidemiological, diagnostic, and therapeutic point of views. About the epidemiological profile, heart valve diseases associated to degenerative processes have become the most frequent pathologies. 1 Regarding the diagnosis, remarkable pro-gresses in imaging techniques allowed a more focused selection of candidates and an optimal timing for surgical correction. In such a context, echocardiography is considered the gold standard for diagnosis of heart valve diseases. 2,3 The best therapeutic approach for their treatment is based on surgical valve replacement by mechanical or biological prosthesis implantation. 1 Although the use of prosthesis is able to guarantee an adequate performance, this approach is characterized by short-term durability, because of structural deterioration, dysfunction, and degeneration processes of prosthesis, also complicated by hemorrhagic events due to a lifelong anticoagulant therapy. 4 All these disadvantages often cause the need of reoperation. 5,6 Based on such considerations, the heart valve tissue engineering (HVTE) applies specific principles to the development of substitutes able to restore, maintain, and improve sick or damaged cardiovascular structures. [6][7][8][9] Current literature evidences the need to identify efficient sources of both ideal scaffolds and cells. Synthetic scaffolds have many advantages in terms of biocompatibility and availability, but they are made of a non-natural matrix, are expensive, potentially immunogenic, and may induce inflammation and toxic degeneration. [10][11][12] Therefore, the use of a biological matrix, able to stimulate a prolonged and positive interaction with adherent cells, appears to be the most suitable solution. Concerning cell sources, many cell types are available for tissue engineering applications, 6 and adult stem/progenitor cells seem to be the best-suited candidates in such a context; they are characterized by low risk for tumorigenesis, no evidences of side effects, and the absence of ethical issues. 13 Among adult stem/progenitor cells, mesenchymal stem cells (MSCs), isolated from different adult tissues, show strong regenerative potential and negative immunomodulatory abilities. 14 In particular, Wharton's jelly, the mucoid connective tissue composing the umbilical cord (UC), is emerging as an interesting source of MSCs (WJ-MSCs), since WJ-MSCs are easily available, stable for several passages, and able to differentiate along the endothelial lineage. 15,16 Moving from these evidences, the present study evaluated the ability of WJ-MSCs and human umbilical vein endothelial cells (HUVECs) 4 to reconstitute an efficient valve endothelium by using a decellularized porcine scaffold as a substratum for the neoendothelium growth.

Materials and Methods
Porcine cardiac valves The present study was approved by the local ethical committee. Six porcine hearts were obtained from ''Di Tomo Oliviero'' slaughterhouse (Chieti, Italy), under veterinary supervision; the material was free from pre-existent cardiac damages. Twelve semilunar porcine cardiac valves (36 heart valve cusps) were obtained under sterile conditions. To evaluate the best protocol for decellularization and reendothelializa-tion processes, either entire porcine heart valves or single porcine heart valve cusps were used.

WJ-MSCs isolation and culture
Human UCs were used to isolate MSCs from cordon matrix. After Wharton's jelly harvesting, the tissue was crumbled, washed, and centrifuged at 250 g for 5 min. The pellet was suspended in Dulbecco's modified Eagle's medium (DMEM; Gibco, Paisley, Scotland) added with 2 mg/dL of collagenase IV (Sigma-Aldrich) and placed in a continuous flutter at 37°C for 16 h. After the enzymatic digestion, the specimen was washed and added with a solution containing 2.5% trypsin-EDTA (Gibco) for 30 min at 37°C. Cell suspension was obtained and the medium was changed every 3-4 days before reaching the confluence; when in subconfluence, cells were detached using trypsin-EDTA 0.05% for 5 min at 37°C, counted, and recultured at a density of 3000 cells/cm 2 . 15 WJ-MSCs endothelial differentiation WJ-MSCs were incubated for 15 days in EGM-2 Bullet-Kit (Lonza, Walkersville, MD) added by 18% of fetal bovine serum (FBS; Gibco). Endothelial phenotype was confirmed through flow cytometry and confocal microscopy.
Cusps were recellularized for 15 days by HUVECs (sixth passage) or WJ-MSCs previously differentiated for 15 days in EGM-2 (eighth passage); in both cases, cells were seeded at the concentration of 1 · 10 6 cells/ mL. The culture medium was changed every 48-72 h.
Heart valve cusps immunocytochemical analysis Confocal microscopy analyses. Cusps (before or after the decellularization/reendothelialization processes) were fixed by 4% paraformaldehyde (Merck, Darmstadt, Germany) for 45 min and nuclei were stained by 0.05 lL/mL 4¢,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) or by DRAQ5 (1:250; Biostatus Limited, Leicestershire, United Kingdom). Cups were stained by anti-CD146-PECy7 (1:20), anti-CD144 or by anti-CD31 (1:40 dilution; BD) CD144 and CD31 were stained by an anti-mouse Alexa Fluor 546 (1:100 dilution; Life Technologies, Carlsbad, CA). Images were acquired using a confocal microscope (LSM510 META; Carl Zeiss, Oberkochen, Germany), equipped with a reverse microscope (Axiovert 200) and a Plan Neofluar 40 · NA 1.3 OIL lens. The excitation was obtained with an Argon laser line (488 nm) and a HeNe laser line (543 and 633 nm). The emission was collected through a primary dichroic HydroTech filter (HTF) of 488/543/633 nm and separated with META array, selecting a range from 680 to 809 nm, while FITC emission was recorded selecting a band pass (BP) filter of 505-550 nm. DRAQ5 emission was recorded through a conventional photomultiplier using a longpass filter (650 nm) and Alexa Fluor 546 through a band pass (BP) filter of 560-615 nm. To prevent overlaps in fluorescence emissions, images were acquired sequentially. 24 For the acquisition of DAPI-stained cusp pictures, a fluorescence microscope (50iEclipse; Nikon, Shinjuku, Japan) was used, and images were acquired with a Cool-SNAPcf digital chargecoupled device camera (PhotoMetrics, Huntington Beach, CA). Digital acquisition, processing, and analysis of fluorescence were performed by Meta Image Series 7.5 (MetaMorph, Metafluor, MetaVue) software obtained from Molecular Devices (LLC, Sunnyvale, CA). 25,26 Multiphoton microscope analyses. Cusps (before or after the decellularization/reendothelialization processes) were fixed by 4% paraformaldehyde for 45 min and stained by 0.05 lL/mL DAPI and the Cell Membrane Labeling Kit PKH26 (1:250 dilution; Sigma-Aldrich). Images were acquired using a Zeiss LSM 7 MP multiphoton microscope (Carl Zeiss, Jena, Germany), equipped with upright microscope Axio-Examiner.Z1 and an objective W-Plan-Apo 20 · /1.0 NA VIS-IR DIC. The two photon excitation was obtained using a Ti:Sapphire Laser Coheren Camaleon Vision II in mode locked (Coherent, Inc., Santa Clara, CA). Excitations were fixed at 730 or 980 nm for DAPI or PKH26, respectively. Fluorescent emission was collected using an NDD detector set in transmission mode using an SP 485 nm filter (DAPI staining) or with a BiG detector in reflection mode equipped with BP 420-475 nm for PKH26.

Valves and cusps decellularization
To decellularize heart cusps, two different detergent solutions, already reported in the literature, were compared: one composed of 1% SDS + 0.05% NaN 3 , 4 and another containing 1% Triton X-100. 17,27 To prove the efficacy of the decellularization process, detergenttreated valves were observed by optical microscopy. Figure 1A-c evidences the complete suppression of the porcine endothelium by fluorescence microscopy since nuclei, pointed out by DAPI staining (blue) on the native cusps (Fig. 1A-a), disappeared when decellularized cusps were observed (Fig. 1A-c). In the green channel, the absence of cell nuclei (Fig. 1A-d) underlined the autofluorescence of the decellularized matrix; such a green autofluorescence disappeared when porcine valve are covered by the native endothelium ( Fig. 1Ab). Data were confirmed by multiphoton microscopy analyses, showing the absence of the cellular component on the scaffold, after staining the decellularized valves with PKH26 (red) and DAPI (blue) (Fig. 1B-d-f); the native porcine endothelium was used as positive control for nuclei and membrane staining (Fig. 1B-a-c). Of note, the absence of PKH26-positive elements on decellularized cusps revealed that nor cells neither cellular fragments (which may induce immune reactions) were evidenced.
Both aforementioned detergents gave similar results in terms of cell removal when compared to the morphology of the freshly harvested valve and cusps ( Fig.  2A, D, respectively). Anyway, the decellularizing solution containing 1% SDS and 0.05% NaN 3 determined a better maintenance of the valve structure ( Fig. 2B) with respect to the corresponding Triton-treated specimens (Fig. 2B, C). For this reason, all the following experiments were performed by using only this solution. In any case, cusps were obtained and cultured (Fig. 2E).
Both aortic and pulmonary valves underwent the decellularization process; results demonstrated that the aortic scaffold better resist to the detergent treatment with respect to the pulmonary one (data not shown). Therefore, we decided to use only aortic roots for the following experiments.

WJ-MSCs endothelial differentiation
After 15 days of culture under endothelial conditions, WJ-MSCs were stained for typical endothelial markers and observed by confocal microscopy. As shown, endothelial-differentiated WJ-MSC expressed both CD31 and CD144 (Fig. 4a, b).

Cusps recellularization
Recellularization was performed culturing single decellularized cusps as substratum for differentiated-WJ-MSCs and HUVECs growth. After 15 days of culture, cusps recellularization was analyzed by fluorescence microscopy; data showed that both WJ-MSCs and HUVECs recolonized the cusps as demonstrated by the presence of nucleated adherent cells on the matrix (Fig. 5). In particular, WJ-MSCs and HUVECs, recellularizing the matrix, displayed characteristic patterns: a whirling cell growth for WJ-MSCs (Fig. 5A) and a uniform distribution for HUVECs (Fig. 5B) were observed. In both cases, cells retained their proliferative capacities; as shown in Figure 5C, the typical metaphase pattern (red arrow) suggested that cells went on dividing once kept in touch with the porcine matrix. Furthermore, the confocal microscopy analysis highlighted that both cell types, when growing on the porcine scaffold, were able to reconstitute a continuous cellular monolayer about 20 lm thick (Fig. 6A), establishing a close contact with the matrix fibers, as if they intercalate among them, as shown by the 3D reconstruction with the isometric projection (Fig. 6B). In any case, both HUVECs and differentiated-WJ-MSCs retained endothelial features when grown on the porcine scaffolds (data not shown).

Discussion
The HVTE constitutes a promising approach to overcome the limitations related to the current surgical treatment of heart valve diseases, nowadays based on the implantation of biosynthetic or mechanical prosthesis. 6 In this context, the reconstitution of functional, durable, and biological heart valve structures requires a deep evaluation of their basic elements (both matrix and cells).
In the present study, a natural scaffold (successfully obtained by decellularizing porcine heart valves) was used, demonstrating that it was able to positively interact with the cell compartment. Natural scaffolds provide bioactive components, ligands, and molecules (collagen, elastin, and proteoglycans), 27 which are probably responsible for their ability, demonstrated here to induce cells to both adhere to the substratum and maintain their proliferative capacities. Ideally, the biological scaffold does not induce toxic degeneration and immunogenic or thrombogenic events, typical reactions often determined by synthetic polymer materials. 4,18,19 For these reasons, it is likely that porcine scaffolds may contribute to the reconstitution of a long-term durable substitute.
The cell component represents another relevant element in the field of HVTE since it has been reported that the in vivo reimplantation of decellularized biological scaffolds induced their fast mechanical deterioration and degeneration. 28,29 In the same way, cell sources need to be carefully chosen to guarantee both a long-term durability of the substitute and a low incidence of adverse reactions; therefore, WJ-MSCs seem to be ideal candidates in such a context. Our results strongly indicated that WJ-MSCs, differentiated along the endothelial lineage, as well as HUVECs (used as positive control), were able to adhere and proliferate when induced to interact with the porcine matrix, reconstituting a neoendothelium layer, characterized by typical endothelial features. The evidenced WJ-MSCs proliferative capacities, when in contact with a natural substratum, let us to hypothesize that they could reconstitute a pluristratified functional endothelium. Of note, differentiated WJ-MSCs demonstrated the ability to express typical endothelial adhesion molecules that normally do not characterize the mesenchymal phenotype; this suggests the tendency of MSCs to differentiate along the endothelial lineage. Anyway, further experiments will be needed to ascertain the maintenance of WJ-MSCs adherence on the scaffold in dynamic conditions. This evaluation may allow to translate the use of these valves in the clinical practice. In this context, it must be highlighted that WJ-MSCs are characterized by broad immunosuppressive properties. Indeed, it has been established that WJ-MSCs are able to inhibit lymphocyte proliferation and their soluble factor secretions. In particular, it has been shown that leukocyte IFN-c production is reduced by WJ-MSCs 14 ; TGF-b1, released by WJ-MSCs, inhibits T-lymphocyte activation and proliferation. 30 Furthermore, WJ-MSCs downregulate many surface markers of monocyte-derived dendritic cells involved in inflam-matory responses (i.e., CD1a, CD14, and the T costimulation complex CD86/CD80). 31,32 For these reasons, the use of WJ-MSCs may also determine a low risk of a graft versus host disease development. Moreover, in the perspective of engineered and neoendothelialized cardiac valve regeneration, it must be underlined that it is possible to cryopreserve autologous WJ-MSCs at childbirth, therefore making them available in any moment of the life of the patient.
Altogether, these evidences introduce a novel idea: the ''custom-made heart valve prosthesis,'' which could real fit on the patient who needs valve substitution. If from one side, this concept could mean a ready availability of the prosthesis, on the other side, it could reduce the incidence of reinterventions, leading to a better quality of life for patients.