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Ann Thorac Surg 2009;87:1196-1203. doi:10.1016/j.athoracsur.2008.12.074
© 2009 The Society of Thoracic Surgeons
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Original Articles: Adult Cardiac

Cell Delivery: Intramyocardial Injections or Epicardial Deposition? A Head-to-Head Comparison

Hadhami Hamdi, MSca, Akira Furuta, MDa, Valérie Bellamya, Alain Bel, MDb, Etienne Puymirat, MDa, Séverine Peyrard, MScc, Onnik Agbulut, PhDd, Philippe Menasché, MD, PhDe,*

a INSERM U 633, Laboratory of Surgical Research, Paris, France
b Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery; Ecole de Chirurgie, Paris, France
c Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Epidemiology and Clinical Research Unit; INSERM CIE4, Paris, France
d Laboratoire de Stress et Pathologies du Cytosquelette, EA300, Université Paris Diderot, Paris, France
e Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery; Université Paris Descartes; INSERM U 633, Paris, France

Accepted for publication December 24, 2008.

* Address correspondence to Dr Menasché, Department of Cardiovascular Surgery, Hôpital Européen Georges Pompidou, 20, rue Leblanc, Paris, 75015, France (Email: philippe.menasche{at}egp.aphp.fr).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background: Multiple needle-based injections of cells in the myocardium are associated with a low engraftment rate, which may limit the benefits of the procedure. This study used skeletal myoblasts to perform a head-to-head comparison of conventional injections with epicardial deposition of scaffold-embedded cells.

Methods: Four weeks after ligation-induced myocardial infarction, 40 rats were randomly allocated to receive intramyocardial injections of 5 million human skeletal myoblasts or control medium or to have the infarcted area covered with either a bilayer myoblast cell sheet prepared from a fibrin-coated culture plate or a myoblast-seeded collagen sponge (Gelfoam; Pharmacia & Upjohn, Kalamazoo, MI). End points, assessed after 1 month, included left ventricular function blindly measured by echocardiography, quantification of cell engraftment by quantitative real-time polymerase chain reaction and immunostaining, histologic assessment of fibrosis and angiogenesis, and tissue levels of host-specific angiogenic and antifibrotic cytokines.

Results: Compared with control medium- or myoblast-injected hearts, those receiving the two cell constructs demonstrated the highest recoveries of left ventricular function (p = 0.004 versus controls). Both myoblast cell sheets and myoblast-seeded Gelfoam sponges also resulted in significantly greater angiogenesis compared with controls. The Gelfoam group was associated with the best outcome with regard to the number of engrafted donor cells (p = 0.03 versus myoblasts) and the reduction of fibrosis (p = 0.02 and p = 0.04 versus the control and myoblast groups, respectively).

Conclusions: Compared with injections, delivery of myoblasts in a construct overlaying the infarcted area is associated with better graft functionality, possibly because of maintenance of improved cell patterning. The cell-seeded Gelfoam construct was found to feature a user-friendly, reproducible, and atraumatic technique.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
During the last decade, the accumulating experience with cardiac cell therapy has allowed us to recognize that the outcome of the procedure was not only dependent on the cell type but also on the methods used for transferring cells and ensuring their optimal engraftment [1]. Consequently, basic research on cell biology has been increasingly paralleled by more technology-oriented studies aimed at optimizing graft delivery and survival. In the setting of surgical applications, this has led us to consider non–injection-based techniques entailing the epicardial coverage of the infarct area by cellularized scaffolds [2–4] with the hope that these patches would reduce mechanical leakage of the donor cells and promote their appropriate patterning with respect to cell-to-cell and cell-to-extracellular matrix contacts, thereby reducing cell death that occurs when anchorage-dependent cells are detached from the extracellular matrix [5]. So far, however, most of these studies have failed to provide a head-to-head comparison of these new approaches with the conventional needle-based injection technique. The present study was designed to address this issue by comparatively assessing, in a rat model of myocardial infarction, the effects of multiple skeletal myoblast injections with those of two epicardially delivered constructs, of which one was made of a scaffold-free myoblast cell sheet and the other of a myoblast-seeded gelatin sponge.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
All experiments were performed in accordance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources, Commission on Life Science, National Research Council, and published by the National Academy Press, revised 1996.

Cell Cultures
Primary muscle cell cultures were prepared from human biopsies taken during orthopaedic operations after institutional approval. The general methodology has already been described [6] and, in brief, entailed digestion of the biopsy by collagenase and trypsin followed by culture in a myogenic-specific medium based on a modification of MCDB120 supplemented with fetal bovine serum, basic fibroblast growth factor, dexamethasone, penicillin, and streptomycin. After expansion until passage 5, aliquots of cells were cryopreserved until the time of use. They were then thawed, resuspended for 4 days in the same medium, and collected by trypsinization.

Preparation of Myoblast Cell Sheets
Biodegradable polymerized fibrin-coated dishes were prepared as described previously [7]. Briefly, a mixture of human fibrinogen (90 mg), thrombin (4 U), aprotinin (3,000 U), and calcium chloride (3.52 mg) was diluted in 16 mL of saline solution, and 0.3 mL of this solution was spread onto 35-mm culture dishes (Corning, Acton, MA). Polymerization was achieved after 2 hours at room temperature. Myoblasts were then plated on the dishes at a density of 6.25 x 104/cm2 in 2.6 mL of a special medium supplemented with 20% fetal bovine serum, 5.0 ng/mL basic fibroblast growth factor, 5.0 ng/mL dexamethasone, and antibiotics (penicillin, streptomycin, and amphotericin). The cell-coated dishes were incubated at 37°C in a humidified atmosphere, and myoblast cell sheets were obtained 9 days later, after the fibrin layer had been gradually degraded by the proteases secreted from the cultured cells. At the time of transplantation, the cell sheets were detached from the plates with a cell scraper. Two of them were overlaid and preincubated to allow stabilization for 30 minutes. To avoid them from being damaged, the stacked cell sheets were mounted onto a collagen film (CLF-01 Koken, Tokyo, Japan), which allowed their easy delivery to the host heart. Immediately after transplantation, the collagen film was peeled off the myoblast cell sheets, which adhered to the epicardial surface of the infarcted area.

Five nontransplanted myoblast sheets were trypsinized and assessed for viability by trypan blue exclusion at five different times (0, 15, 30, 60, and 120 minutes). Other sheets were either fixed in cold methanol (–20°C, 10 minutes) and then assessed for the presence of myogenic cells by desmin immunostaining (1:100; DakoCytomation, Glostrup, Denmark) after 9 days of plating or tested for cell proliferation by Ki-67 (1:30; Abcam, Cambridge, MA) at days 1 (negative control), 9, and 21 after plating.

Preparation of Cell-Seeded Gelfoam Sponges
Gelfoam sheets (Pharmacia & Upjohn, Kalamazoo, MI) were cut into 2.5 x 1.0 x 0.5 cm3 blocks, hydrated in phosphate-buffered saline for 30 minutes, and seeded with human myoblasts at a density of 0.8 x 105 (6 drops, one in each corner and two in the central part of the Gelfoam piece). The cell-loaded Gelfoam blocks were then incubated for 3 hours at 37°C with 5 % CO2 before transfer into a six-well plate containing 3 to 5 mL of medium supplemented with 20% fetal bovine serum, 5.0 ng/mL basic fibroblast growth factor, and 1% combined antibiotics in each well. The medium was changed every 72 hours, and the cell-seeded scaffolds were grafted after 3 weeks. Control nontransplanted sponges were digested with collagenase (0.5 mg/mL, Type 1A; Sigma, St. Louis, MO), and after centrifugation the cell pellet was assessed for viability by trypan blue exclusion. Other sponges were frozen and subsequently cryosectioned for visualization of myogenic cells by immunostaining against desmin.

Myocardial Infarction Model
Female Wistar rats weighing 250 g were anesthetized with 2% to 3% isoflurane (Baxter, Maurepas, France). After tracheal intubation, mechanical ventilation (Alphalab; Minerve, Esternay, France) was set at a rate of 70/min and with a 2-mL average insufflation volume. The heart was approached through a left thoracotomy, and a myocardial infarction was created by permanent ligation of the left coronary artery with a 7-0 polypropylene snare (Ethicon, Somerville, NJ).

Four weeks after creation of infarction, rats underwent a baseline echocardiographic assessment of left ventricular (LV) function, and only those with an ejection fraction between 0.20 and 0.45 were selected for the trial. After a median sternotomy, these animals were randomly allocated to receive intramyocardial injections of culture medium (controls, n = 10) or skeletal myoblasts (5 x 106, n = 10) or to undergo epicardial deposition of either a 9-day cultured myoblast cell bilayer sheet (n = 10) or a myoblast-seeded Gelfoam sponge (n = 10) overlaying the infarcted area. All injections consisted of a 150-µL volume delivered in three sites in the core and at the borders of the scar by using a 29-gauge needle. Although the myoblast sheets spontaneously adhered to the surface of the heart, the Gelfoam sponges had to be secured by two 7-0 polypropylene stitches. Immunosuppressive therapy, consisting of one daily 10 mg/kg subcutaneous shot of cyclosporin A, was started on the day of transplantation and continued thereafter until sacrifice.

Assessment of Left Ventricular Function
Pretransplantation and posttransplantation cardiac function was evaluated before transplantation and 1 month thereafter by transthoracic echocardiography (Sequoia 516, equipped with a 15-MHz transducer; Siemens, Mountain View, CA) in animals sedated with 2% isoflurane. Parasternal long- and short-axis views were obtained with both M-mode and two-dimensional images. Left ventricular end-diastolic surface, LV end-systolic surface, LV end-diastolic length, and LV end-systolic length were measured on parasternal long-axis views with two-dimensional images. Volumes were calculated as (8/3{pi}) x (surface2/length). Ejection fraction was calculated as (LV end-diastolic volume – LV end-systolic volume)/LV end-systolic volume. All measurements were made in triplicate and averaged by an investigator blinded to the treatment group.

Quantitative Real-Time Polymerase Chain Reaction
Because the primary objective of this study was to assess engraftment, the majority of hearts (7 of 10 in each group) were processed for quantification of cell numbers by quantitative real-time polymerase chain reaction (qRT-PCR). Total RNA was extracted from rat myocardium using the RNA NOW 60-minute one-step mRNA reagent (Biogentex, Seabrook, TX), reverse-transcribed from an oligo(16)dT with Mu-MLV reverse transcriptase (Invitrogen, Cergy, France), and qRT-PCR was performed with SYBR Green mix (Roche, Mannheim, Germany) on a Light Cycler FastStart DNA Master Plus (Roche). The human-specific primer sequences (HuG6PDH-R) were (forward/reverse) 5'-ATGGGGAAGGTGAAGGTCGGAG-3'/5'-TCGCCCCACTTGATTTTGCAGG- 3'.

To assess whether delivery of donor cells had triggered host-associated angiogenic or antifibrotic pathways, qRT-PCR was also used to quantify transcripts for rat-specific vascular endothelial growth factor-A (VEGF-A) and hepatocyte growth factor (HGF). The primer sequences were (forward/reverse) VEGF-A: 5'-tcaccaaagccagcacatag-3'/5'-ttgaccctttccctttcctc-3'; and HGF: 5'-TATTTACGGCTGGGGCTACA3'/5'-ACGACCAGGAACAATGACAC-3'.

The messenger RNA expression levels of target genes were normalized to hypoxanthine-guanine phosphoribosyltransferase and transcription factor II D signals as housekeeping genes, and all experiments were performed in triplicate.

Cytokine Array
Frozen heart samples were placed into an ice-cold homogenization buffer containing (in mmol/L) 20 Tris (pH 7.6), 250 NaCl, 3 EDTA, 3 EGTA, 0.5% nonyl phenoxylpolyethoxylethanol 40, 2 dithiothreitol, 10 Na-orthovanadate, 10 NaF, 10 glycerophosphate, and an antiprotease inhibitor cocktail (Sigma). Samples were homogenized and then centrifuged at 12,000g for 30 minutes at 0°C. Protein concentration was measured by the method of Bradford using bovine serum albumin as a standard. Cytokine antibody arrays were carried out according to the manufacturer's instructions (Ray Biotech, Norcross, GA) using 200 µg of protein per sample. The system allows us to screen multiple mediators, including cytokine-induced neutrophil chemoattractants 2 and 3, ciliary neurotrophic factor, fractalkine, LPS-induced CXC chemokine, leptin, monocyte chemotactic protein 1, macrophage inflammatory protein 3{alpha}, nerve growth factor β, granulocyte macrophage-colony stimulating factor, interferon-{gamma}, tissue inhibitor of metalloproteinase 1, tumor necrosis factor {alpha}, VEGF, and interleukins 1a, 1b, 4, 6, and 10. The expression of stroma-derived factor 1{alpha} and placenta growth factor was also tested separately. The intensity of the different spots was determined using a densitometric software (Scion Image, NIH, Bethesda, MD).

Western Blot Analysis
Tissue lysates (40 µg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis before electrophoretic transfer onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). Western-blot analysis was carried out using anti-rat VEGF-A (Santa Cruz Technology, Santa Cruz, CA) and anti-rat HGF (Euromedex, Souffelweyersheim, France) antibodies. Antibody reacting bands were visualized after development with peroxidase-conjugated secondary antibodies (Pierce Biotechnology, Rockford, IL) and a chemiluminescent detection system (ECL-Plus; GE Healthcare, Chalfont St. Giles, UK). Bands were quantified by densitometric software (Scion Image, NIH).

Histologic and Immunohistochemical Assessment
After the last echocardiographic assessment, three hearts in each group were separated in two halves by a short-axis section through the midportion of the infarcted area. The blocks were immediately fixed in Tissue-TeK Optimal Temperature Cutting medium (Sakura, Torrance, CA) and frozen at –180°C in liquid nitrogen until they were sliced into 7-µm-thick cryosections using an ultramicrotome (LM 1850; Leica, Wetzlar, Germany). Forty fields (10 per heart) randomly spanning the entire infarct area were analyzed with a microscope (Leica DMIL) equipped with a digital camera (Qicam; Qimaging, Burnaby, BC, Canada). Hematoxylin and eosin staining was used to delineate the area of infarction. The presence of human cells was detected by immunofluorescence using an antibody directed against a specific nuclear human protein (lamin A/C; Novocastra, A Menarini Diagnostics, Rungis, France) at a magnification x40. Myogenic cells, macrophages, and T lymphocytes were identified by immunostaining using antibodies against fast skeletal myosin (clone My32, 1:200; Sigma), CD68 (clone ED-1, 1:100; Serotec, Oxford, UK), and CD3 (1:75; DakoCytomation, Trappes, France), respectively. The proteins were revealed using fluorescein isothiocyanate- or Texas red-conjugated secondary antibodies. The presence of these different cells in the myocardial tissue was then graded according to a semiquantitative score where 1+, 2+, and 3+ corresponded to minimal, moderate, and massive infiltration, respectively. Endothelial cells were immunostained with a rat-specific antibody (RECA, clone HIS52, 1/30; Serotec, Dusseldorf, Germany) conjugated with a biotinylated anti-mouse IgG secondary antibody (Vector, Burlingame, CA). Angiogenesis was then computed by counting RECA-positive cells at a magnification of x10. The extent of fibrosis was assessed by Sirius red staining at a magnification x5. Digital images were then processed with Metamorph software (Universal Imaging Corporation, Downington, PA), and fibrosis was expressed as the ratio between the area of scar tissue to the LV area. Nuclei were counterstained with hematoxylin or 4', 6-diamidino-2-phenylindole.

Data Analysis
Data are summarized using median (minimum; maximum) or mean (±1 standard deviation) values. The changes in LV function variables (LV ejection fraction, LV end-diastolic volume, LV end-systolic volume) were compared among treatment groups using a parametric analysis of covariance with the presacrifice measurement as outcome and the pretransplantation measurement and treatment groups as covariates. The mean numbers of vessels per square millimeter and the mean percentages of fibrosis were compared among groups using a mixed model for clustered continuous data, taking into account the intraheart correlation (as multiple slices were used for a single heart) or a nonparametric Wilcoxon rank sum text incorporating the cluster effect. The percentages of fibrosis were log-transformed to satisfy hypotheses required for the parametric model. Statistical significance was set at the 5% threshold, and all statistical analyses were performed with the SAS statistical software version 9.1 (Cary, NC.).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
In Vitro Data
Proliferation of myoblasts in the cell sheet, as assessed by Ki-67 immunostaining, peaked at day 9 but sharply dropped thereafter, hence the choice to graft the sheets at the 9-day time point. At this time, the sheets had become so sticky that they could not be trypsinized for cell counting, but the growth kinetics suggested that each of them contained approximately 1 million myoblasts so that a twofold greater number of cells were delivered to the heart once two sheets had been piled one on top of the other. Proliferation of myoblasts within the Gelfoam sponge was important. Three of these scaffolds, initially seeded with 80,000 cells, were tested for the presence of myoblasts by desmin staining and, after 3 weeks, the number of cells was found to have increased to approximately 3 x 106. In all groups, pretransplantation cell viability was greater than 90%.

Functional Outcome
Baseline LV ejection fraction was not different among the four groups and averaged (mean ± standard deviation) 0.32% ± 0.7%, 0.30% ± 0.9%, 0.32% ± 0.7%, and 0.29% ± 0.5% in the control, myoblast, Gelfoam, and cell sheet groups, respectively (p = 0.75). However, the patterns of changes were then divergent as LV function deteriorated compared with baseline values in control hearts, stabilized in the myoblast group, and increased in the two cell construct groups (Fig 1). After adjustment for baseline values, the posttransplantation LV ejection fraction (mean and 95% confidence interval) was thus found to be higher, although not significantly, in the myoblast group compared with controls (estimated difference of means: 7.2% [–0.6 to 15.0], p = 0.08), whereas the best recoveries were yielded by the Gelfoam (Pharmacia & Upjohn) and cell sheet groups (estimated difference of means with controls, 10.3% [2.5 to 18.0] and 10.3% [2.5 to 18.2], respectively, both p = 0.004). Left ventricular end-diastolic and end-systolic volumes were not significantly different among the four groups (data not shown).


Figure 1
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  		Fig 1. Baseline left ventricular ejection fraction (EF) in all groups and percentage of change 1 month after transplantation. Data are given as mean ± standard deviation.

 
Cell Engraftment
Seven hearts were processed for quantification of cell engraftment by polymerase chain reaction. Human cells could be detected in two, two, and five of them in the myoblast, cell sheet, and Gelfoam groups, respectively. The DNA concentration (median [min, max]), as determined by qRT-PCR, was 0.00 ng/µL [0.00, 2.60], 0.00 ng/µL [0.00, 88.00], and 6.20 ng/µL [0.00, 17.40] in the myoblast, cell sheet, and Gelfoam groups, respectively (p = 0.03 for Gelfoam versus myoblasts), but we failed to find a significant correlation between the amount of residual DNA in each heart and the corresponding functional outcome. Immunostaining against the fast skeletal muscle marker My32 (Fig 2) showed that in the two scaffold-treated groups, myogenic cells remained confined within the epicardial construct and had not migrated into the infarcted area. In keeping with the polymerase chain reaction data, the presence of these cells was minimal (1+) in the cell sheet group and greater (2+ or 3+) in the Gelfoam group.


Figure 2
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  		Fig 2. Detection of human cells in myoblast-seeded collagen sponge (Gelfoam; Pharmacia & Upjohn, Kalamazoo, MI) before (A) and after (B–F) implantation in infarcted myocardium. (A–C) Immunofluorescence staining shows the presence of desmin-positive cells in the myoblast-seeded collagen sponge 3 weeks after cell culture (A). One month after implantation in infarcted myocardium, immunofluorescence shows the presence of many human cells identified by their positive staining for the human lamin A/C (B, D); some of them express the adult fast myosin heavy-chain marker (My32) and have differentiated into multinucleated myotubes (C). (D–F) Grafted cell expressing the human marker lamin A/C (D) and the myogenic marker My32 (E). Picture F represents the merged image. Nuclei are stained blue by 4', 6-diamidino-2-phenylindole. Bar = 75 µm (A); 125 µm (B); 25 µm (C); 10 µm (D–F).

 
Angiogenesis
There was a greater degree of angiogenesis in the two scaffold-treated groups. Thus, the median (min, max) number of vessels per square millimeter was 190 (187, 199), 304 (221, 327), 520 (330, 528), and 426 (397, 448) in the control, myoblast, Gelfoam, and cell sheet groups, respectively (Gelfoam versus controls, nonadjusted p = 0.03; cell sheet versus controls, nonadjusted p = 0.02).

Fibrosis
The extent of fibrosis, expressed as a percentage of the infarcted area, featured grossly similar patterns. The median (min, max) percentage was 66% (61%, 73%), 61% (51%, 79%), 36% (34%, 44%), and 43% (28%, 94%) in the control, myoblast, Gelfoam, and cell sheet groups, respectively. Only the difference between the Gelfoam and the control and myoblast groups reached statistical significance (nonadjusted p = 0.02 and p = 0.04, respectively). Immunostaining against ED1 showed that the infiltration of the two scaffolds and the underlying infarcted area by macrophages was overall moderate and tended to be still more limited in the cell sheet group. The infiltration by CD3 was minimal in the three cell-treated groups, thereby reflecting the efficacy of the immunosuppression regimen.

Expression of Cytokines
There was no significant difference among groups with regard to the expression of mRNA for VEGF or HGF, as determined by qRT-PCR, or the expression of the tissue levels of the corresponding proteins, as determined by Western blot analyses (data not shown). Likewise, levels of the multiple mediators screened by the cytokine array system as well as those of stroma-derived factor 1{alpha} and placenta growth factor failed to show significant between-group differences.


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
A consistent finding of cell therapy studies is the very low rate of sustained cell engraftment, which can drop as low as 1% of the initial number of donor cells a few weeks after transplantation [8]. Taking into account that the cardiomyocyte deficit resulting from an infarction large enough to cause heart failure is in the range of 1 billion cells [9], one cannot realistically expect a clinically meaningful benefit from such a tiny number of residual donor cells, particularly in the case of skeletal myoblasts for which a clear dose-effect relationship has been documented [10]. This low rate of engraftment is initially caused by a mechanical leakage of cells [11–13] and subsequently worsened by an interplay of biologic factors that include inflammation, ischemia owing to poor vascularization of the injected areas, and apoptosis subsequent to detachment of anchorage-dependent cells from their extracellular matrix, so-called anoikis [14]. The recognition of these contributing factors provides a rationale for embedding cells into three-dimensional biodegradable scaffolds that might better preserve cell survival and proliferation compared with proteolysis followed by injection of isolated cells. Furthermore, the epicardial delivery of these scaffolds avoids the drawbacks associated with multiple needle-based injections, which include (1) a washout of cells through channel leakage and the vascular system, (2) an inhomogeneous distribution of cells, (3) the disruption of the extracellular matrix and the subsequent loss of signals that modulate cell survival, differentiation, and patterning, (4) the lack of reproducibility, and (5) a potential proarrhythmic risk attributable to the conduction blocks created by the clusters of cells interspersed in the disrupted myocardial tissue [8]. On the basis of these grounds, two conceptually different constructs, cell sheets and collagen scaffolds, were tested in the present study.

Scaffold-free cell sheets can be manufactured onto temperature-sensitive [15] or, like in our experiments, fibrin-coated culture dishes [7]. In both settings, two detached cell sheets can be piled one on top of the other as beyond this number, the thickness of the construct may prevent adequate oxygenation of the outer layers. The advantages of this approach include (1) the lack of any foreign material, (2) the maintenance of intercellular connections through anchorage of the cells to a self-secreted extracellular matrix, and (3) possibly a mechanical cyclic strain-induced orientation of the layered cells toward a cardiomyogenic phenotype [16]. The main limitation of the cell sheets is their fragility, which complicates manipulations at the time of transplantation. In the present study, the myoblast cell sheet group yielded better results than the conventional injection technique with regard to preservation of LV function, angiogenesis, and to a lesser extent, limitation of fibrosis. These data are consistent with those that have been reported with myoblast cell sheets prepared onto temperature-sensitive dishes, both in ischemic [17] and nonischemic [18] cardiomyopathy models. However, an unexpected observation was that this superior outcome did not correlate with a greater rate of engraftment as there were only very few residual donor cells detectable after 1 month. The reason for this massive graft loss is unclear, but it is noteworthy that, so far, a successful use of cell sheets prepared onto fibrin-coated dishes has only been reported with rat neonatal cardiomyocytes [19]. Of note, in this study, there was evidence for bidirectional electrical communication between the host heart and the cell sheet without inducible arrhythmias by a programmed stimulation protocol, which strengthens the concept that replacement of intramyocardial injections by epicardial cell deposition may increase the safety of stem cell delivery. However, one cannot exclude that myoblast culture on fibrin-coated dishes is less effective than when it is done on temperature-sensitive plates because stem cell–biomaterial interactions have been shown to be cell-specific [20]. Furthermore, whereas myoblasts were cultured for 9 days onto the fibrin-coated dishes, the incubation time with temperature-sensitive dishes is shortened to 1 to 2 days. This temporal difference may also affect the patterning of intercellular connections and the associated anchorage-dependent survival signaling. Indeed, temperature-sensitive dishes look particularly appealing because their surface characteristics seem to accommodate a large variety of cells for the manufacturing of cell sheets.

Collagen-based constructs have already been shown to exert powerful angiogenic effects, regardless of whether collagen was used in a liquid form in conjunction with cells [21] or a cell-free solid scaffold [22]. In the present study, we selected to seed myoblasts onto Gelfoam, a three-dimensional gelatin matrix with a honeycomb-like structure, because this material features excellent handling characteristics and allows adequate cell growth kinetics, as demonstrated by the large increase in the myoblast number within 3 weeks of incubation inside the patch. Previous studies have established that Gelfoam sponges were effective vehicles for delivering mesenchymal stem cells in cartilage regeneration therapy [23], endothelial cells for control of vascular repair after arterial injury [24], and cardiomyoblasts for preserving function of heterotopically transplanted hearts [3]. Interestingly, it has also been shown that endothelial cells embedded in Gelfoam were protected from both allogeneic and xenogeneic immune rejection [25], but this property was not tested in this study because the parallel use of human myoblasts (in suspension or layered as cell sheets) required in any event an immunosuppressive therapy. Our data confirm that incorporation of skeletal myoblasts in Gelfoam is an effective means of increasing cell engraftment and correspondingly improved LV function, increased angiogenesis, and reduced fibrosis. Although we did not include a group receiving a cell-free patch, it is unlikely that these beneficial effects could be attributed to the matrix alone as epicardial deposition of an acellular collagen sponge has failed, in a similar model, to provide any benefit [4], probably because this material is too soft to mechanically scaffold the infarct area and prevent its remodeling. Of note, the remaining cells were confined within the patch without evidence for penetration within the myocardium. This may not be such an issue with skeletal myoblasts, which, because of their lineage-restriction, are in any event unable to convert into cardiomyocytes and thus establish electromechanical connections with the host cardiac cells [26]. Previous studies, however, have reported that epicardially delivered rat neonatal cardiomyocytes were able to foster such connections [19], and patch-embedded mesenchymal stem cells have also been shown to invade the underlying infarcted area [2]. Thus, the extent to which the phenotype of the grafted cells along with their migratory capacity from the supporting patch may affect the patterns of scar recolonization remains to be investigated.

Technical factors related to the optimal cell-seeding density for each substrate (collagen sponge or fibrin-coated dish) and to the material-specific growth characteristics of the cells precluded us from matching the ultimate number of myoblasts that were delivered in each group. Clearly, however, this number was smaller in the cell sheet and Gelfoam groups, and the finding that hearts in these two groups yet demonstrated superior outcomes compared with those injected intramyocardially strengthens the conclusion about the efficacy of epicardial cell delivery. Interestingly, although the two construct-treated groups yielded equivalent functional recoveries, they markedly differed with regard to the number of residual cells, which were much more abundant in the Gelfoam-treated hearts than in those covered with the myoblast sheets. Similarly, a cardiac patch made of mesenchymal stem cells embedded in collagen has been shown to improve postinfarction myocardial remodeling despite the lack of detectable cells 4 weeks after patch implantation [4]. Although the xenogeneic setting of the present study may have aggravated graft loss despite immunosuppression, this effect would be expected to have been equally balanced among all groups. The reason for the discrepancy in cell numbers between the two patch groups remains unclear but could be related to the fact that the three-dimensional collagen scaffold onto which myoblasts were incorporated was more conducive to their survival and proliferation than the thin bilayer cell sheet. Regardless of the causes for this difference in cell numbers, these data support the assumption that transplanted cells do not primarily act by physically replacing dead cardiomyocytes but rather activate host-associated endogenous pathways that subsequently remain operative on their own even though the trigger cells have disappeared. This paracrine mode of action is thought to account for the common discrepancy between the functional benefits of cell transplantation and the scarcity of long-term donor cell engraftment [27]. It is supported by the previous observations that delivery of human endothelial progenitors in nude mice [28] or transplantation of mouse myoblasts in swine [29] results in an upregulation of host-associated cytokines and growth factors. Likewise, human mesenchymal stem cells embedded into a collagen patch epicardially applied onto rat hearts have been shown to increase the number of native recipient-specific myofibroblasts [4]. Recently, additional compelling evidence for the predominant involvement of cell-released mediators has been offered by a study in which the sole injection of the cell-derived conditioned medium was sufficient to trigger cardioprotective effects in a porcine model of myocardial infarction [30]. However, in the present study, our combinatorial approach looking at both mRNA and protein expression (by two different techniques) failed to demonstrate the upregulation of various recipient-specific cytokines, particularly VEGF and HGF. A possible explanation is that these mediators are relatively short-lived, which could have prevented an increase in their tissue levels from being detectable after 1 month whereas the events they have triggered (increased angiogenesis, reduced fibrosis) are still present. Indeed, in the study by Cho and coworkers [28], which has demonstrated the activation of host-associated signaling pathways by engrafted cells, the follow-up was limited to 14 days. Likewise, in experiments showing that the myoblast cell sheet resulted in higher tissue levels of VEGF and HGF than injections of the same myogenic cells, polymerase chain reaction was performed 7 days after transplantation [17]. In line with these findings, in the study in which a mouse myoblast cell line was injected into swine hearts, angiogenesis was found to be significantly higher than in controls after 1 month whereas at the same time, transcripts for porcine VEGF were absent or only minimally expressed [29]. Of note, the present data suggest that the paracrine effects largely documented for mesenchymal stem cells [31–33] are also relevant to skeletal myoblasts, which is in line with the previous observation that epicardially applied myoblast cell sheets release cardioprotective signaling molecules more effectively than when these cells are injected intramyocardially [17]. Altogether, these results are consistent with the report by Ebelt and coworkers [34] and our own findings [35] that myoblasts release a blend of cardioactive growth factors and cytokines favorably modulating cardiomyocyte survival, inflammation, matrix composition, and scar formation.

In conclusion, these data suggest that the benefits of cell transplantation can be potentiated by an epicardial deposition of the cells, as opposed to their usual intramyocardial injection. The advantages of this mode of delivery, which likely extend beyond the sole use of skeletal myoblasts, could make cell therapy both more effective and more user-friendly in that a preshaped cellularized patch made of a clinically usable material can be expeditiously and reproducibly deposited onto the surface of the heart during a surgical open chest procedure.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This research was supported in part by a grant from the LeDucq Foundation (Cardiac Progenitors Transatlantic Alliance network 04 CVD 04).


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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