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Ann Thorac Surg 2008;86:132-141. doi:10.1016/j.athoracsur.2008.02.074
© 2008 The Society of Thoracic Surgeons
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Thoracic Surgery Directors Association Award

Stem Cell–Derived, Tissue-Engineered Pulmonary Artery Augmentation Patches In Vivo

Bret A. Mettler, MDa, Virna L. Sales, MDa,b, Chaz L. Stucken, BAa, Vesa Anttila, MDa, Karen Mendelson, MAc, Joyce Bischoff, PhDb,d, John E. Mayer, Jr, MDa,b,*

a Department of Cardiovascular Surgery, Children's Hospital Boston, Boston, Massachusetts
d Department of Surgery, Vascular Biology Program, Children's Hospital Boston, Boston, Massachusetts
b Harvard Medical School, Boston, Massachusetts
c Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts

Accepted for publication February 21, 2008.

* Address correspondence to Dr Mayer, Department of Cardiovascular Surgery, Children's Hospital Boston, 300 Longwood Ave, Boston, MA 02115 (Email: john.mayer{at}cardio.chboston.org).

Presented at the Forty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 29–31, 2007. Winner of the Thoracic Surgery Directors Association Resident Research Award.


Pediatric cardiac surgery: The Annals of Thoracic Surgery CME Program is located online at http://cme.ctsnetjournals.org. To take the CME activity related to this article, you must have either an STS member or an individual non-member subscription to the journal.

 

   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
 References
 
Background: Reconstruction of the right ventricular outflow tract is a frequently encountered component of many congenital cardiac repairs. We sought to tissue engineer pulmonary artery augmentation patches from retrovirally labeled endothelial progenitor and mesenchymal stem cells and determine the persistence of the seeded cells in vivo.

Methods: Autologous ovine endothelial progenitor and mesenchymal stem cells were labeled with a retroviral vector encoding green and red fluorescent proteins, coseeded onto biopolymers, and cultured for 5 days. The tissue-engineered patches were implanted into the main pulmonary artery with 1, 2, 4, and 6 week in vivo maturation (n = 8). In vivo evaluation included ultrasonography and angiography, with preimplant and explanted specimens evaluated using histologic examination and immunofluorescence.

Results: Echocardiography at each time demonstrated laminar pulmonary artery flow without a pressure gradient across the replaced segment. Pulmonary angiography did not exhibit stenosis or aneurysmal change. Gross appearance of all explanted patches showed progressive tissue formation with increased length of time in vivo. Retrovirally labeled cellular persistence was 96%, 82%, 85%, and 66% at 1, 2, 4, and 6 weeks after implantation, respectively. Early in the in vivo remodeling period, the number of green fluorescent protein–positive endothelial progenitor cells was 1.6 fold greater than the red fluorescent protein–positive mesenchymal stem cells. As in vivo remodeling continued, red fluorescent protein–expressing mesenchymal stem cells were expressed 1.2 to 1.7 times that of the green fluorescent protein–positive endothelial progenitor cells.

Conclusions: The data demonstrate the successful creation of an anatomically functional, autologous tissue-engineered pulmonary artery using coseeded progenitor cell sources. Labeled implanted stem cells persisted in the engineered construct, suggesting that in vitro seeding is necessary to engineer tissue. This study demonstrates an effective method to track multiple cell types after implantation.


The Thoracic Surgery Directors Association (TSDA) Resident Research Award was established in 1990 to encourage resident research in cardiothoracic surgery. Abstracts submitted to The Society of Thoracic Surgeons (STS) Program Committee representing research performed by residents were forwarded to the TSDA to be considered for this award. The abstracts were selected by the TSDA Executive Committee consisting of Jeffrey Gold, MD, President, John Brown, MD, President-Elect, John Calhoon, MD, Secretary/Treasurer, Douglas Mathisen, MD, Immediate Past President, George Hicks, MD, Councilor-at-Large, Bartley Griffith, MD, Councilor-at-Large, and Leslie Kohman, MD, Councilor-at-Large.

In 2007 there were two recipients of the TSDA Resident Award: Robert A. Meguid, MD, a resident of Stephen Yang, MD, at Johns Hopkins Medical Institutions and Brett A. Mettler, MD, a resident of John E. Mayer, Jr, MD, at Children's Hospital Boston. The awards were presented at the STS 43rd Annual Meeting in San Diego, CA. Each TSDA Award recipient received a monetary award of $1000.

The TSDA makes this award annually. The resident authors of the selected studies are recognized at the STS meeting.

 

Reconstruction of the right ventricular outflow tract is an important procedure in the surgical treatment of congenital heart disease. In the majority of patients, reconstruction can be achieved using a bioprosthetic or synthetic material, with limitations being lack of growth potential, limited size variability, and the requirement for anticoagulation in some patients [1]. An autologous tissue-engineered construct capable of cellular division and tissue growth could compensate for the functional shortcomings of currently available options used in congenital cardiac surgery.

Tissue engineering uses a combination of cells, bioengineered materials, and biochemical cues to improve or replace anatomic and physiologic tissue functions. Initial tissue-engineered cardiovascular structures used autologous cells from the intima and media of blood vessels [2, 3], with pursuant experiments involving cellular coseeding [4]. In an attempt to use less invasive cell sources, tissue-engineered structures have used umbilical cord endothelial cells [5], ovine endothelial progenitor cells (EPC) [6], and bone marrow–derived mesenchymal stem cells (MSC) [7]. A tissue-engineered construct coseeded with both circulating endothelial and pluripotential mesenchymal cellular components has yet to be evaluated.

A persistent question in cardiovascular tissue engineering is whether cell populations initially placed on the scaffold persist after in vivo implantation [8] or whether tissue remodeling using nonseeded cell sources repopulate these implants. Proposed sources for in vivo remodeling include cells recruited from circulating and bone marrow–derived progenitor cells or cellular in-migration from the surrounding native cardiovascular tissue [9]. The persistence of the implanted cells and their exact role in tissue histogenesis is difficult to determine.

Our hypothesis is that tissue-engineered pulmonary artery augmentation patches derived from autologous circulating EPC and bone marrow–derived MSC will form a tissue substitute suitable for in vivo implantation. Furthermore, we evaluated the persistence of the implanted cells using retroviral vectors expressing green fluorescent protein (GFP) and red fluorescent protein (RFP) in EPC and MSC, respectively.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Discussion
 Acknowledgments
 References
 
Animal care and experimental procedures were approved by the Animal Care Committee of the Children's Hospital Boston.

Isolation, Characterization, and Culture of Mesenchymal Stem Cells and Endothelial Progenitor Cells
Female lambs (n = 8) were used for all experiments. Mesenchymal stem cells were isolated from bone marrow and cultured as previously described [7, 10]. Culture medium was composed of Dulbecco's Modified Eagle's Medium (Invitrogen, Carlsbad, CA), 20% fetal bovine serum, and 1% antibiotic-antimycotic solution (GIBCO). The isolated cells were shown to be multipotential by differentiation along both osteogenic and adipogenic lineages [10], with differentiation assessed using von Kossa and oil-red O staining, respectively. Endothelial progenitor cells were isolated from peripheral blood as previously described [11]. Culture medium was composed of EBM-2 media supplemented with EGM-2 SingleQuots (Clonetics) without hydrocortisone, antibiotics, and 20% fetal bovine serum. Cells were assayed for CD-31 (1:1000, Santa Cruz) and von Willebrand factor (1:500, DAKO) using immunofluorescence and Western immunoblotting. Both cell phenotypes were characterized before retroviral introduction.

Retroviral Transduction of Endothelial Progenitor and Mesenchymal Stem Cells
Retroviral vectors were constructed as previously described [12]. The viral medium was collected and cells were transduced with 1 x 109 particles of high-titer retrovirus. The EPC were transduced with GFP [13], and the MSC were transduced with RFP [14]. Both transduced and nontransduced cells were characterized before and after retroviral transduction.

Tissue-Engineered Augmentation Patch Maturation
Nonwoven polyglycolic acid biopolymer (Albany Int) was cut into 1- x 2-cm segments coated with 1% wt/vol poly-4-hydroxybutyrate tetrahydrofuran solution (Tepha Inc) [8]. Cells were simultaneously coseeded onto scaffolds at a cell density of 2 x 105 MSC/cm2 and 2 x 106 EPC/cm2. The autologous patches were implanted into the main pulmonary artery of the same animal 10 weeks after initial cell isolation. A segment of pulmonary artery was excised and closed with the augmentation patch. All animals underwent direct echocardiography and pulmonary angiography at the time of sacrifice. Animals were sacrificed at 1, 2, 4, and 6 weeks after implantation.

Analysis of the Engineered Tissue
In vitro and in vivo tissue engineered patches as well as resected native pulmonary artery were stained with hematoxylin and eosin. Immunofluorescence detection of GFP-positive and RFP-positive cells used mouse anti-GFP monoclonal antibody (1:100, Molecular Probes) and rabbit anti-HcRed (1:50, Clontech Laboratories), followed by Alexa 488-conjugated goat anti-mouse immunoglobulin G (1:500) and Alexa 594 goat anti-rabbit immunoglobulin G (1:1000; Molecular Probes). Nuclear counterstaining was performed with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes).

Quantitative tissue analysis was performed by counting GFP- and RFP-expressing cells after 1, 2, 4, and 6 weeks' duration and comparing the cellular persistence in relationship to the preimplanted tissue-engineered patches. Total cellularity was described as the number of DAPI-expressing nuclei with cells counted in three high-power fields by a blinded observer. Both GFP-positive and RFP-positive labeled cells were expressed as a percentage of the total DAPI-positive cells.

Apoptosis and Proliferation Studies
Detection of proliferating GFP-positive and RFP-positive cells was performed using mouse monoclonal anti-Ki-67 (1:28, Abcam) antibodies, chicken anti-GFP antibodies (1:38, Abcam) and rabbit anti-HcRed (1:25, Clontech Laboratories) antibodies followed by Alexa 488-conjugated goat anti-mouse (1:1000) and Alexa 594 goat anti-rabbit and chicken immunoglobulin G (1:500, Molecular Probes). Nuclear counterstaining was performed with DAPI. Slides were analyzed using fluorescence microscopy. Apoptotic cells were identified using terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick end–labeling (TUNEL). Overall proliferation and apoptosis data for each cell type were determined by counting the number of Ki-67–positive and TUNEL-expressing cells colocalized with the GFP- and RFP-expressing cells. Proliferating and apoptotic cell numbers were expressed as a percentage of the total number of DAPI-positive cells.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Discussion
 Acknowledgments
 References
 
In Vitro Characterization of Endothelial Progenitor and Mesenchymal Stem Cells
The EPC expressed CD31 on the cell-cell membrane borders (Fig 1A) and von Willebrand factor in cytoplasmic granules (Fig 1B). Mesenchymal stem cell isolates initially grew in small, rounded clusters and expressed {alpha}-smooth muscle actin (Fig 1C), similar to vascular smooth muscle cells (Fig 1D). Multipotency of the MSC was demonstrated by inducing cells to differentiate along osteogenic and adipogenic lineages. Alkaline phosphatase expression, characteristic of osteogenic differentiation, was detected after 2 weeks in osteogenic-specific media (Fig 1E). Cytoplasmic lipid vacuoles accumulated after 2 weeks of culture in adipogenic media (Fig 1F). Before patch seeding, fluorescent protein gene transfer was achieved in greater than 99% of the cultured precursor cells (Figs 2A, 2B). After 5 days of incubation, hematoxylin and eosin staining showed cells were more abundant in the superficial layers (Fig 2C) and DAPI expression colocalized with the GFP and RFP protein marker in the preimplant tissue-engineered constructs (Fig 2D).


Figure 1
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  		Fig 1. Immunofluorescent detection of antibodies to ovine endothelial markers CD31 (A) and von Willebrand factor (B). Ovine mesenchymal stem cells (C) are immunoreactive to {alpha}-smooth muscle actin similar to blood vessel–derived smooth muscle cells (D). Mesenchymal stem cells were differentiated in osteogenic and adipogenic culture-specific media. Mesenchymal stem cells cultured in osteogenic media (E) stain positive for alkaline phosphatase whereas those cultured in adipogenic media (F) show accumulation of lipid vacuoles when stained with oil-red O. (A–D, x400; E, F, x100.)

 

Figure 2
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  		Fig 2. In vitro culture of labeled endothelial progenitor cells with green fluorescent protein (GFP; A) and labeled mesenchymal stem cells with red fluorescent protein (RFP; B). (C) In vitro tissue-engineered pulmonary artery augmentation patch after 5 days in laminar flow system stained with hematoxylin and eosin. (D) The same section of pulmonary artery augmentation patch under fluorescent microscopy (fluorescein isothiocyanate–Texas red) demonstrating nuclear labeling with 4',6-diamidino-2-phenylindole (DAPI; blue) colocalized for RFP-labeled mesenchymal stem cells (red arrows) and GFP-labeled endothelial progenitor cells (green arrows) before in vivo implantation. (A, x100; B, x200; C, x40; D, x400.)

 
In Vivo
All animals survived implantation with somatic growth during the in vivo period averaging 1.5 kg/week. Before sacrifice at 1, 2, 4, and 6 weeks, echocardiography demonstrated laminar pulmonary artery flow without a pressure gradient across the replaced segment. Pulmonary angiography did not exhibit stenosis or aneurysmal change. Gross appearance of all explanted patches showed progressive tissue formation with increased length of time in vivo (Figs 3A–3D). The luminal surface at 1 week contained thrombus (Fig 3A, 3E) and progressed to a smooth pulmonary artery surface with in vivo maturation.


Figure 3
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  		Fig 3. Representative patches of progressive tissue formation during a 6-week in vivo maturation period. (A) Thrombus formation is seen after 1 week in vivo with minimal evidence of tissue formation. At 2 (B), 4 (C), and 6 weeks (D), the patches show a progressive smooth endothelial surface with increased tissue formation. After 6 weeks in vivo, the tissue-engineered patch grossly assimilates native pulmonary artery. (E) Light microscopy hematoxylin and eosin section of tissue-engineered pulmonary artery augmentation patch after 7 days in vivo. The region below the line represents the tissue-engineered patch. (PA = native pulmonary artery; + = area of fibrin clot on the luminal surface.) (F) After 6 weeks of in vivo maturation, tissue-engineered patch architecture assimilates architecture of native pulmonary artery (PA). The area to the left of the line represents the native pulmonary artery. (* = anastomotic suture site.) (E, x100; F, x40.)

 
In Vivo Tissue Expression of Retroviral-Labeled Endothelial Progenitor and Mesenchymal Stem–Derived Cells
Using immunofluorescence, GFP-positive EPC and RFP-positive MSC were localized in the implanted tissue-engineered patches at all times. Retrovirally labeled cells were absent in the adjacent native pulmonary artery. Comparing total cell number with cells expressing GFP and RFP, labeling effectiveness was 95% before implantation. Retrovirally labeled cellular persistence was 96%, 82%, 85%, and 66% at 1, 2, 4, and 6 weeks after implantation, respectively (Fig 4A). The EPC expressing GFP composed 58% of the cells in the patch after 1 week of in vivo remodeling and decreased to 32% at 6 weeks (Fig 4B). The MSC had a peak expression of 51% during the second week and decreased to 33% at 6 weeks (Fig 4C). Early in the in vivo remodeling period, the number of GFP-positive EPC was 1.6 fold greater than the RFP-positive MSC. As in vivo remodeling continued, RFP-expressing MSC were expressed 1.2 to 1.7 times that of the GPC-positive EPC (Fig 5).


Figure 4
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  		Fig 4. Preimplant in vitro seeded tissue-engineered patches compared with the same autologous patch after 1, 2, 4, and 6 weeks of in vivo maturation comparing total tissue cellularity, green fluorescent protein (GFP), and red fluorescent protein (RFP) expression when compared with the number of 4',6-diamidino-2-phenylindole (DAPI)-expressing cells. (A) Cell seeding is held constant in all autologous in vitro patches used as in vivo controls. As length of time in vivo increases, the number of cells expressing a fluorescent protein marker decreases. (B) Comparison of endothelial progenitor cells (EPC) expressing GFP before implant versus cohort described in vivo maturation. In the preimplant patch, tissue cellularity averages 47% of GFP-positive EPC across all times. After 1 week in vivo, the tissue-engineered patch contains the largest percentage of GFP-positive EPC (58%) and decreases as length in vivo increases. (C) Similar comparison of mesenchymal stem cells (MSC) expressing RFP with 48% of the preimplant tissue-engineered patch composition containing RFP-positive MSC. The maximal number of cells (51%) identified with the RFP marker occurs at 2 weeks of in vivo maturation.

 

Figure 5
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  		Fig 5. (A) In vivo tissue-engineered patch. Green fluorescent protein (GFP) and red fluorescent protein (RFP) expression in the tissue-engineered patches after 1, 2, 4, and 6 weeks of in vivo maturation. At 1 week, cellularity was predominately GFP-labeled endothelial progenitor cells (EPC) with a more homogeneous distribution as length in vivo increases. (B) Immunofluorescent labeling of the tissue-engineered patch at 1, 2, and 6 weeks. Tissue-engineered patches have maximal endothelial progenitor cell expression at 1 week and maximal mesenchymal stem cell (MSC) expression at 2 weeks. At 6 weeks, the population of nucleated cells has less expression of assayed retroviral markers. (B, x400.) (green arrows = GFP-positive EPC, red arrows = RFP-positive MSC, blue = 4',6-diamidino-2-phenylindole (DAPI)-expressing cells.)

 
In Vivo Proliferation and Apoptosis for Tissue-Engineered Patch
Tissue sections were stained for Ki-67 and TUNEL immunoreactivity. Ki-67 is expressed by cycling cells and provides a quantitative estimate of the number undergoing mitosis [15]. The highest percentage of proliferating EPC expressing GFP was 58% after 1 week; peak MSC expressing RFP was 51% after 2 weeks (Figs 6A–6C). At 6 weeks, we observed a greater percentage of TUNEL-positive cells, a smaller percentage of proliferating cells, and the fewest number of retrovirally labeled cells in the tissue-engineered patch (Figs 4A, 6C, 6D).


Figure 6
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  		Fig 6. (A) Representative tissue-engineered patch assayed for proliferation (green) and green fluorescent protein (GFP)–labeled endothelial progenitor cells (EPC) (red). Nucleated cells stained with 4',6-diamidino-2-phenylindole (DAPl) (blue). Single arrows denote cells expressing both Ki-67 and GFP, indicative of proliferating GFP–labeled EPC (x400). (B) Mesenchymal stem cell (MSC) proliferation evaluated using proliferation (green). Cells were also assayed for red fluorescent protein (RFP) expression of the MSC (red). DAPl was used to stain all cellular nuclei. Single arrows demarcate proliferating MSC. At all time points (data not shown), a greater degree of proliferation is seen near the tissue-engineered construct–native pulmonary artery border (x400). (C) The percentage of EPC and MSC proliferating was determined by counting the number of cells expressing both Ki-67 and either GFP or RFP and dividing by the number of DAPl–positive cells. After 1 week in vivo, 56% of GFP–positive EPC are proliferating, decreasing to 28% at 6 weeks. RFP–positive MSC show maximal proliferation at 2 weeks (51%), decreasing to 28% at 6 weeks. (D) Terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick end–labeling (TUNEL) staining for apoptosis increased with increased time in vivo. At 1 week, 2.7% of cellular elements were TUNEL-positive, increasing to 10.5% at 6 weeks.

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Discussion
 Acknowledgments
 References
 
Adult progenitor cells have independently been used for tissue-engineered cardiovascular structures [6, 7]. This study used both cell types concomitantly to tissue engineer a cardiovascular structure. The model presented represents an autologous, cardiovascular augmentation patch derived from EPC and MSC. With the short duration of in vivo maturation, it is unclear whether tissue formation will be suitable for long-term implantation, yet early time points are encouraging and supportive of future in vivo experimentation.

Potential sources of cell populations in engineered cardiovascular tissue include the arterial-derived cells initially seeded onto the scaffold [8], circulating cells that localize to the construct [6, 7, 16], and local migratory cells from the surrounding cardiovascular tissue. In this study, we attempted to determine whether the implanted cells seeded onto the polymer scaffolds persisted in vivo. We used in vitro labeling of progenitor cells with retroviral vectors before cellular coseeding. Using fluorescence microscopy, we confirmed that the EPC expressed GFP and MSC expressed RFP before scaffold seeding. After 6 weeks of in vivo maturation, we found labeled GFP-positive and RFP-positive cells in the engineered cardiovascular tissue. This finding confirmed successful labeling of the progenitor cells and persistence of protein expression in vivo and suggests that our engineered tissues are partially derived from cells that were initially transplanted.

Our results show that the cellular composition at early times within the tissue-engineered scaffolds are derived from in vitro seeding and may be associated with early extracellular matrix production. Published studies have demonstrated that both preimplant and implanted tissue-engineered patches contain extracellular matrix constituents. After 6 weeks in vivo, the seeded patches contained interstitial glycosaminoglycans and collagen [17]. Other work has shown that MSC-seeded pulmonary valve conduits developed progressive tissue formation for 4 months in vivo [16]. Isolated EPC-seeded constructs showed increased cellularity and contained both extracellular matrix proteins and matrix remodeling enzymes [11, 18]. We believe that simultaneous seeding of EPC and MSC in our augmentation patch model contributed to the early tissue formation.

With a longer duration of in vivo maturation, a lower percentage of cells expressed GFP and RFP although the total cell number increased. We presume that cells from other sources are populating the vascular patch. A proposed model would involve recipient cell migration from the adjacent vessel into the tissue-engineered construct. Conversely, we found no evidence of seeded progenitor cell migration from the patch to the adjacent pulmonary artery. However, it is possible that adjacent vessel cellular contribution to tissue formation is associated with vascular remodeling [19–21]. A second model of tissue formation involves circulating progenitor cells incorporating into the tissue-engineered scaffold. It is possible that the initial seeded cell population could provide signaling cues for circulating cell populations [6, 16, 22]. Finally, it may be that the seeded retrovirally labeled cells stopped expressing the fluorescent protein marker. A decreased expression is less plausible in that transgene expression was stable during a 6-month period of in vitro cell culture. Detection of the fluorescent protein marker in engineered tissues requires the use of antibodies for enhancement [12, 20, 23]. As the construct becomes more tissuelike with an increased amount of extracellular matrix protein deposition, the protein marker becomes more difficult to detect and may falsely lower the number of seeded cells identified.

A debate exists regarding the necessity of cellular preseeding of tissue-engineered constructs. We believe the implanted cells support tissue histogenesis through microenvironmental stimuli that influence cellular division, migration, and tissue formation with protein mediators. Exposure of EPC-seeded engineered tissue to transforming growth factor-β1 simulated phenotypic transdifferentiation to a mesenchymal phenotype with extracellular matrix production [11, 18], suggesting that the seeded cells have intrinsic characteristics that may influence the cellular content of the engineered tissue. Using a combination of progenitor stem cells provides a more diverse cellular substrate to stimulate generating tissue. Several studies have demonstrated that seeding multiple cell phenotypes results in a thicker graft with more extensive angiogenesis [24]. Because several cell types are present in autologous vascular conduits, seeding with a mixture of cells may be more beneficial than seeding with a purified cell source [25]. We chose EPC and MSC as they have the potential to differentiate into a variety of cell phenotypes with a greater capacity for self-renewal.

The cellular contribution to the tissue-engineered construct was initially represented by a preponderance of EPC with an EPC-to-MSC ratio of 10:1. After 6 weeks of in vivo maturation, the ratio of EPC to MSC was equal. Early in the in vivo maturation period, the cellular discrepancy may be related to the exponential difference in cell number used to seed the patches. The predominant decrease in the number of GFP-expressing EPC is associated with the formation of a continuous endothelial lining and may be explained by a combination of circulating and local endothelial cells mobilized to establish endothelial integrity. The contribution of endogenous endothelial cells could be assessed with identification of a marker not expressed on engineered tissue. After implantation, the tissue-engineered construct is exposed to hemodynamic forces that may "wash" unattached cells from the patch. Additional investigation evaluating the reticuloendothelial system for fluorescent protein expression is required. An initial increase in the total cell number expressing fluorescent proteins correlates with increased EPC proliferation and low apoptosis. After 6 weeks of in vivo maturation, both cell types showed a 30% to 50% decrease in proliferation and a 75% increase in apoptosis. We postulate that the seeded cells play a role in promoting early engraftment of the tissue-engineered patch through the release of mediators supporting cell mobilization and extracellular matrix formation. Cell-signaling interplay between engineered tissue resident and circulating precursors may contribute to the dynamic process of tissue remodeling and maturation in vivo.

Study Limitations
We have not performed control studies such as nonseeded or singly seeded EPC or MSC vascular patches and believe these studies are necessary to evaluate initial cellularization of the tissue-engineered construct. The results using tissue-engineered vascular patches support others' results using in vitro seeding of progenitor cells. A previously published study using acellular vascular patches showed aneurysmal dilatation. After 26 weeks in vivo, seeded vascular patches were without aneurysm formation in the pulmonary circulation [8]. A carotid interposition model demonstrated that nonseeded grafts thrombosed within 15 days whereas grafts seeded with EPC remained patent for 130 days [6]. Tissue-engineered heart valves seeded with MSC functioned in vivo for greater than 8 months and remodeled, assimilating a native heart valve containing an organized, extracellular matrix [16]. This suggests that in vitro seeding of MSC is an important component for tissue generation [26]. Shin'oka and associates [27] implanted unseeded control grafts in the inferior vena cava position with an 80% occlusion rate compared with a100% patency of MSC-containing vascular grafts. We believe that the in vitro seeding of cocultured EPC and MSC played a pivotal role in the tissue remodeling observed. Longer-duration studies will be necessary to assess whether the combined coculture effect of both cell types will provide the best substrate for tissue-engineered vascular grafts.

Early thrombus formation after implantation requires additional investigation into the functional status of the endothelium of the engineered tissues. Previous studies on explanted tissue-engineered grafts using EPC revealed a confluent monolayer of endothelial cells without surface thrombus after 15 days. The same grafts were patent after 20 weeks in vivo and exhibited nitric oxide–mediated vascular relaxation [6]. Shin'oka and associates [27] reported that MSC-derived tissue-engineered vascular grafts acquired a functioning endothelial surface, defined by nitric oxide production, after 14 days in vivo and maintained patency up to 32 months. Based on these observations, our tissue-engineered grafts are unlikely to have a functional endothelium at the time of implantation. The presence of surface thrombus on the tissue-engineered construct requires the use of antiplatelet therapy in the early postoperative period.

Conclusion
The present study describes an anatomically functional, autologous tissue-engineered pulmonary artery augmentation patch constructed in vitro from circulating EPC and pluripotential bone marrow–derived MSC. The engineered vascular grafts functioned in vivo for up to 6 weeks and grossly began to resemble the structure of the native pulmonary artery. Furthermore, we demonstrated that the presence of labeled implanted cells persisted from the in vitro seeding of the tissue-engineered cardiovascular patch after 6 weeks of tissue maturation. The persistence of the labeled cells suggests not only that these cells are critical to the tissue generation process but that in vitro cell seeding is necessary to engineer tissue. Finally, this demonstrates a reproducible model for labeling multiple cell types in tissue-engineered constructs.


   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Discussion
Acknowledgments
 References
 
DR CARL L. BACKER (Chicago, IL): What about that thrombus on the patch, does that make you nervous? Would that be something you would want to treat clinically or is that part of the healing process and necessary for the patch to form the way it does?

DR METTLER: Our initial suspicion is that this is part of the healing process. All these animals, while not discussed, are maintained on aspirin in the immediate postoperative period. But until they have a functional endothelial lining, which the functionality of the endothelium was not investigated in this study but has been investigated by Sunjay Kaushal who I believe is in the audience at this time, but until the endothelium is evaluated, it's hard to say that there's a functional endothelium.

Of interest is that as our time in vivo increased, we did have an endothelial lining.

DR JOHN W. BROWN (Indianapolis, IN): Land-breaking work.

Next question, obviously this is a static patch. How about making a mono cusp out of this material?

And will motion of the patch improve the functionality of the graft?

DR METTLER: That's an excellent question. As you know from Dr Mayer's lab, they have tissue engineered heart valves primarily from arterial-derived cells in the initial experimental experience followed by using mesenchymal stem cells. This is the first experience where we've put both cellular constituents of endothelial origin and mesenchymal origin into a cardiovascular tissue-engineered structure.

I certainly think that the next step is to perform a tissue-engineered heart valve, but there are many other operative challenges that are involved using ovine cardiopulmonary bypass to undergo survival.

DR TARA KARAMLOU (Toronto, Ontario, Canada): Just a couple quick questions. One, I missed the particulars of the animal model. I'm not sure if you mentioned it, what species you were using?

Second, you looked at stenosis and some other hemodynamic variables, and it seems to me that 6 weeks might be a bit premature to look at some of those end points.

And thirdly, if someone is in the audience who could just make a quick comment about endothelial cell functionality in this study. In other words, were they able to synthesize nitric oxide, for instance? If you could give us just a one-line answer, that would be great.

DR METTLER: Certainly. The answer to your first question is it's an ovine model.

I think the second question is the functionality in regard to the endothelium in tissue-engineered constructs in regard to endothelial progenitor cells is in a Nature paper by Kaushal et al., where they tissue engineered carotid vessels. In that paper, they show that there is nitric oxide production of the endothelium, and I'll reference you to that paper.

And your last question was in regard to?

DR KARAMLOU: I just wondered if you were looking at stenosis, et cetera, and—

DR METTLER: Excuse me, yes. I agree that stenosis is very early in this time frame, but to be able to compare long-term in vivo studies and have a frame of reference, this data is critical. It certainly is something, though, that will be more applicable in long-term in vivo studies.

DR SUNJAY KAUSHAL (Ann Arbor, MI): I had two questions for you.

One was how big was the patch, which you implanted in the RVOT (right ventricular outflow tract)?

And the second question was at 6 weeks when you looked at the retroviral-infected EPCs (endothelial progenitor cells) and MSCs (mesenchymal stem cells), did you do H and E (hematoxylin and eosin) staining of those patches to determine if there's any other cell types in there?

DR METTLER: Sure, both good questions. The patches were 2 by 1 cm, so they were small.

And the answer to second question is we did do H and E staining of these. There has shown to be early endothelial cell in migration likely from the native pulmonary artery. But it's unclear at this time as on the cell surface, there is green fluorescent protein cells which are likely cells that we've labeled in the tissue-engineered construct.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
References
 
This work was supported by NIH grants HL-60463 and HL-68816-01, Tepha/NIST grant NANB2H305, and CIMIT (J.E.M.); by NRSA/NIBI grant F32 EB003353-01 (B.A.M.); and by AHA 0635620T (V.L.S.). The authors acknowledge contributions of David Martin, PhD (Tepha Inc), Marsha Moses, PhD (Children's Hospital Boston), Elena Aikawa, MD, PhD (Massachusetts General Hospital), and Frederick J. Schoen, MD, PhD (Brigham and Women's Hospital).


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
 References
 

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