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Ann Thorac Surg 2004;78:1409-1417
© 2004 The Society of Thoracic Surgeons
a Department of Cardiovascular Surgery and Cardiology, Haut-Lévêque Hospital, Pessac, France
b National Health Institute and Medical Research, Pessac, and University of Bordeaux 2 - Victor Segalen, Bordeaux, Cedex, France
Accepted for publication December 29, 2003.
* Address reprint requests to Dr Barandon, Cardiovascular Surgery Department, Haut-Lévêque Hospital, 33604 Pessac, France
lesbarandon{at}wanadoo.fr
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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METHODS: Myocardial infarction was induced cryogenically in backcrossed ROSA 26 transgenic x C57BL/6J mice (n = 75). Thirty days later, surviving mice (n = 69) were randomized to sham treatment (rethoracotomy only; n = 11), patch only treatment (n = 29), or patch + BMC treatment (n = 29). Abdominal muscle patches were harvested from donor littermates not expressing the ß-galactosidase reporter gene and sutured on the epicardium directly above the infarct zone. Patch only–treated mice received uncoated patches. Patch + BMC–treated mice received patches coated with 5 x 106 ß-galactosidase-expressing BMCs embedded in a collagen-rich three-dimensional matrix.
RESULTS: Mortality rate was 52% after muscle patch implantation. Bone marrow cells were able to migrate from muscle patch into the infarct zone, as demonstrated by ß-galactosidase immunostaining, and ultimately constituted 8% of all cells in scar tissue (mean ± standard deviation, 219 ± 111/mm2). Angiogenesis and cell survival in the scar were improved by patch + BMC treatment. Left ventricular geometry and cardiac function were improved by patch treatment, with or without BMC, although the effects were stronger after patch + BMC treatment.
CONCLUSIONS: Epicardial deposition of a BMC-coated muscle patch is a promising approach to restoring cardiac function after myocardial infarction.
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Introduction |
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Several promising therapies for the repair of cardiac tissues irreparably damaged by ischemic heart disease and MI have been developed. These therapies include cardiomyoplasty, cell transplantation, and cardiac tissue engineering [2, 3]. Cardiomyoplasty, in which the latissimus dorsi muscle is used to create a left ventricle (LV) wrap [4], was initially greeted by cardiac surgeons with enthusiasm but has been limited in its effectiveness by the problem of muscle degeneration [5]. This problem has been solved to some degree, but not completely, by using electrical stimulation [6] and growth factors [7] to help preserve muscle and to allow long-term LV remodeling and stabilization of cardiac function to occur [8].
Cell transplantation into infarcted areas of the heart has been made possible by improvements in cell biology, selection, and culture [9]. Contractile cells (myoblasts, cardiomyocytes, smooth muscle cells), noncontractile cells (fibroblasts), and progenitor cells (bone marrow, peripheral progenitor cells) have all been used for this purpose [10, 11]. However, several questions remain unanswered, including the most effective cell type to use, the optimal cell dosage, the timing of transplantation, the effects of in situ cell differentiation, and the ability of transplanted cells to become and remain contractile [12–14]. In addition, the cell transplantation approach is limited by poor cell survival and grafting as a result of cell trauma and inflammation, especially when cells are injected directly into the myocardium.
Cardiac tissue engineering has shown much promise. A method of cardiac tissue engineering has already been developed in which cardiac tissue is grown within a three-dimensional scaffold composed of a growth factor–laden, collagen-rich matrix [15]. The scaffold is applied to the epicardium directly above an infarcted myocardial area to improve capillary density and perfusion in the damaged area [16]. However, use of cellular therapy has been limited by the early death of transplanted cells, and scaffolds need to include cells to be used in MI regeneration [17]. To overcome these limitations, we have developed a variation on the three-dimensional scaffold method whereby we introduce bone marrow cells (BMCs) into a three-dimensional collagen matrix and then cover the scaffold with a muscle patch to improve the long-term viability and integrity of the cell-containing matrix. In the study reported here, we tested the feasibility of this combined method of using BMCs coated on an epicardial muscle patch to restore cardiac function in a mouse model of MI.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Mice
All mice used in this study were obtained by backcrossing ROSA 26 transgenic mice (Jackson Laboratory, Bar Harbor, ME), which ubiquitously overexpress the ß-galactosidase reporter gene, and C57BL/6J mice (Jackson Laboratory) for at least eight generations. Only male backcross offspring 9 to 12 weeks old and 26 to 30 g weight were used for this study. All mice received humane care in compliance with the European Convention on Animal Care (L358 to 86/609/EEC). This included being maintained in cages with access to food and water ad libitum. Backcrossed mice positive for the ß-galactosidase reporter gene were used as BMC donors [11, 18]. Only C57BL/6J littermates negative for the ß-galactosidase reporter gene were used as muscle patch donors; this was done to limit potential problems related to histocompatibility.
Myocardial Infarction Model
Myocardial infarction was induced by freeze-thawing (cryoinjury) as previously described [19]. In brief, recipient mice were anesthetized with a mixture of ketamine (2.5 mL/kg) and xylazine (0.8 mL/kg) injected intraperitoneally and then subjected to thoracotomy. A metallic, 1.5-mm-diameter cryoprobe cooled to minus;190°C by immersion in liquid nitrogen was applied to the free wall of the LV twice for 20 seconds each time to induce transmural necrosis and MI. Once MI was induced, the thoracotomy incision was closed in three layers, and the pneumothorax was exsufflated. After intraperitoneal rehydration and warming, mice were revived, returned to their cages, and maintained there as described above for 30 days.
Study Treatments
After MI had been induced by cryoinjury, mice were randomly assigned to one of three treatment groups. One group underwent sham treatment (ie, rethoracotomy and immediate closure) 30 days after MI induction. A second group underwent rethoracotomy and placement of a muscle patch, without BMC, 30 days after MI induction. A third group underwent rethoracotomy and placement of a muscle patch coated with BMCs 30 days after MI induction. All groups of mice were also injected with bromide-deoxyuridine 24 hours before sacrifice for the purpose of measuring the rate of cell proliferation. All mice that survived sham, patch only, or patch + BMC treatment for 15 days were sacrificed.
Isolation and Placement of Muscle Patches
The muscle patches used in patch only–treated or patch + BMC–treated mice were each isolated and prepared as follows. Thirty days after MI induction, a median laparotomy was performed, through which a 1 x 1-cm patch of abdominal muscle was harvested. By using an 8-0 polypropylene purse suture, the muscle patch was cupped so as to be able to fit the LV geometry and, in the case of patch + BMC–treated mice, to form a "tank" in which to deposit and secure BMCs (see below for a detailed description of the deposition procedure).
Isolation, Preparation, and Deposition of Bone Marrow Cells
The BMCs used in patch + BMC-treated mice were obtained from ß-galactosidase-positive donor mice and prepared for epicardial deposition on muscle patches as follows. First, the donor mice were sacrificed by lethal injection of sodium pentobarbital. Then, their femurs were dissected. Next, BMCs were extracted from the femoral bone marrow and purified in Ficoll Paque (Amersham Biosciences, Orsay, France). Expression of ß-galactosidase in the extracted BMCs was demonstrated by X-Gal staining and by ß-galactosidase immunostaining; in our hands, these methods revealed that only approximately 60% of the extracted BMCs were positive for ß-galactosidase (data not shown). Once extracted, BMCs were not subjected to any further culturing; instead, 5 x 106 ß-galactosidase-positive BMCs were immediately mixed into a growth factor–deprived liquid matrix rich in collagen type 1 (100 µL, 200 mg/mL; Becton Dickinson Biosciences, Bedford, MA) that had the peculiar ability to polymerize and harden at 37°C (Fig 1b). This BMC-containing matrix was then deposited within the cupped area of the muscle patch, where it was allowed to polymerize and harden. While the matrix was hardening, the recipient mouse was subjected to rethoracotomy. The cupped, BMC-coated patch was then positioned on the exposed epicardium directly above the MI zone and secured with three sutures (septal, lateral, and apical; Fig 1c). Finally, the rethoracotomy incision was closed. After recovery from surgery, the recipient mouse was treated with oral doxycycline (200 mg/mL) for 1 week to prevent sepsis. (It should be noted that doxycycline was used even with the knowledge that it may affect cardiac remodeling [20].)
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Histologic Analysis
Morphometric Analysis
Immediately after completion of the hemodynamic studies, infarct size was determined in a minimum of 5 mice in each treatment group as described previously [22]. In brief, mice were sacrificed by lethal injection of potassium chloride. Their hearts were fixed by pressure perfusion with a 4% paraformaldehyde solution; this was done to avoid collapse or distension of the LV and to preserve the LV geometry. Next, the heart was cut perpendicular to the longitudinal axis of the LV to create two equal sections. These two sections were embedded in paraffin and cut into serial 7-µm-thick sections, mounted on slides, stained with Masson's trichrome, and finally examined and photographed with a charge-coupled device camera (Nikon, SMZ) connected to an IBM personal computer. Infarct area, LV cavity area, and MI scar thickness were determined manually by a blinded observer and averaged for each treatment group. Infarct size was calculated as the percentage of total LV area that exhibited necrosis.
Immunohistochemical Analysis
All remaining mice in each group were sacrificed by lethal injection of sodium pentobarbital. Their hearts were removed, fixed in methanol, and prepared for immunohistochemical analysis as previously described [23]. To detect transplanted cells (ie, those expressing ß-galactosidase), cardiac tissue specimens were stained with ß-galactosidase antibody (1/2000, Chemicon) and with X-Gal [24]. To detect angiogenic activity, endothelial cells were stained with CD-31 antibody (1/20, Pharmingen). To determine the extent of transplanted cell proliferation, specimens were immunostained with bromide-deoxyuridine antibody (1/20, Harlan). A minimum of 30 random digital photographs were taken at x40 magnification for each mouse specimen analyzed. Positively stained cells were manually counted by a blinded observer, with the help of Sigma Scan Plot software. The number of transplanted cells, capillary density, and cell proliferation per square millimeter were determined and recorded.
Statistical Analysis
All data were expressed as mean ± standard deviation. All analyses were performed using appropriate software (Statview 5.1). Comparisons of continuous variables between two groups were made using one-way analysis of variance and, if a statistically significant difference was observed, a two-sided paired Student's t test. A value of p less than 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Modification of Myocardial Infarction Scar Tissue after Epicardial Deposition of Bone Marrow Cells Beneath a Muscle Patch
Migration of Bone Marrow Cells into Scar Tissue
In patch + BMC–treated mice, a mean of 219 ± 111 ß-galactosidase–positive BMC/mm2 were detected within MI scars 15 days after treatment. This represented 8% of the total cell population of the scar. In contrast, no ß-galactosidase–positive cells were detected in the MI scar in either sham-treated mice or patch only–treated mice (Table 1, Fig 2). Although many transplanted cells were detected in the MI zone, epicardium, and endocardium, most were detected in the ischemic border zone. Few transplanted cells were found in uninjured zones. Together, these findings suggested that the deposition of BMCs directly onto the epicardium between scar and patch allowed their migration into the MI zone.
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Our use of mice ubiquitously expressing retrovirally transduced ß-galactosidase reporter genes (ROSA 26) as BMC donor mice was based on our strategic need to detect transplanted cells after treatment. In this way we could avoid adenovirus infection, which is associated with a very strong inflammatory response that leads to heavy loss of transplanted cells. To reduce the chances of immunologic incompatibility, we also backcrossed ß-galactosidase mice onto receiver mice (C57Bl/6J) and then used the resulting littermates that were negative for expression of the ß-galactosidase gene as receiver mice. In this way we could avoid using immunosuppressive drugs, which are known to modify the healing process after MI [25].
Our decision to use an autologous muscle patch was made for two main reasons. First, we could not use a vascularized latissimus dorsi. It was not possible in mice to perform the microsurgical technique to use a vascularized muscle. Second, we had found in pilot studies that autologous abdominal muscle patches could become easily integrated into—and in some cases even merge with—the epicardium. These advantages were offset somewhat by the tendency of the abdominal muscle patches to become thinner over time because of ischemic complications; however, this thinning was significantly less severe when the patch was coated with BMCs, presumably owing to the evident tropic and degradation-inhibiting effects of the BMCs. To reduce patch degradation even further, we are currently investigating the use of electrical current.
Our choice of day on which to implant the patches (day 30 after MI induction by cryoinjury) was based on several factors. First, we had found in pilot studies that to implant a patch before then was technically difficult. In brief, we had tried to implant patches at several times after infarction, including immediately and at 4, 7, 15, and 30 days after MI. Although implantation immediately or on day 4 after MI was not too technically difficult, the inflammatory response still prevalent at those times caused the loss of many of the transplanted cells and degradation of the patch. On days 7 and 15, the suturing technique was, in our hands, technically difficult to perform because the scar tissue arising from the MI was often adherent to the ribs. This not only made rethoracotomy very difficult, but also resulted in frequent wounding of the infarct zone. By day 30, however, the healing process was complete, which not only provided better conditions under which to attempt rethoracotomy but also fortuitously eliminated confounding factors such as the early inflammatory response that might obscure the real effect of our novel combination therapy.
Our decision to use BMCs as our transplant cells of choice was based on several known features of BMCs, namely, their ability to regenerate infarcted scar tissue [26], to differentiate into endothelial cells and cardiomyocytes in vitro and in vivo [27–29], and to resist ischemia. The last feature is especially important for purposes of cardiac regeneration. In addition, recent clinical trials using BMCs had produced encouraging results [30, 31].
Our strategy of delivering transplanted cells to irreparably damaged tissues by means of epicardial deposition beneath a muscle patch is controversial, mainly because it flies in the face of most experiments with cardiac cell transplantation to date. More often than not, previous cell transplantation studies have involved the injection of cells directly into damaged myocardial tissues. The intramyocardial injection technique is limited, however, by (1) the extensive mechanical trauma to cells (and consequent apoptosis) that can be caused by the syringes and needles that must be used, (2) the limited total number of cells in solution that can be injected, and (3) and the inhospitability of the scar environment to transplanted cells. Thus, in developing our epicardial deposition strategy, we have tried to offset these disadvantages by making it possible to (1) use unrestricted numbers of transplanted cells; (2) deposit cells in a "trauma-free" way that avoids the use of inappropriate devices and avoids immunogenic reactions; (3) place transplanted cells in a favorable environment (ie, collagen) conducive to their survival, proliferation, and migration; and (4) use a homologous muscle patch as a reservoir in which to allow transplanted cells in the matrix to form a stronger, more adherent bond with the patch itself.
As our present results clearly suggest, the relatively large number of BMCs transplanted by means of our novel technique—5 x 106 cells as compared with the usual 1 x 105 cells used in other murine studies [26]—remained viable for at least 15 days after implantation and in that time the cells were able to migrate into and proliferate within the adjacent myocardial scar tissue. Nearly 8% of the transplanted cells were later detected in the scar, a result certainly underestimated because of the low rate (only 60%) at which we were able to identify ß-galactosidase–positive BMCs in our study. Moreover, our strategy significantly improved angiogenesis in scar tissue, as demonstrated by fivefold greater number of new vessels seen in the scar tissue of patch + BMC–treated mice than in either sham-treated or patch only–treated mice. We have hypothesized two possible mechanisms for this improved angiogenic activity. One possibility is the increased secretion of angiogenic factors that are capable of improving endothelial cell formation in scar tissue. The other possibility is BMC transdifferentiation in endothelial cells [11]. We have recently succeeded in demonstrating the transdifferentiation of BMCs into endothelial cells, but we have not been able to demonstrate their transdifferentiation into skeletal muscle. Further experiments are required to explore this possibility.
Together, the present findings indicate that our combination of cell transplantation and cardiomyoplasty (ie, deposition of a BMC-coated abdominal muscle patch on the epicardial surface) was able to reverse the degradation of cardiac function caused by experimentally (cryogenically) induced MI. However, it is not clear whether the therapeutic effect of this combined therapy was caused more by the mechanical modification of LV geometry by the muscle patch, resulting in reduced LV dilation by restriction during diastole [32]; by the migration of BMCs into scar tissue, resulting in increased angiogenesis and cellularity; or by both sets of processes equally. Although we did not analyze blood flow in the infarcted zone of the hearts in our experimental mice, we hypothesize that the increase in capillary density did improve scar perfusion and consequently cardiac function.
In conclusion, our findings in a murine model of MI suggest that a combined therapy of cell transplantation and cardiomyoplasty can restore function to irreparably damaged ischemic LV myocardium both biologically (through the direct or indirect effects of BMCs deposited on the epicardium) and mechanically (through an abdominal muscle patch). Our findings also suggest that it might be possible to maintain transplanted cells in a favorable environment, even while they are in direct contact with an inhospitable epicardial environment, until they can migrate into and thereby increase the viability of both scar and muscle patch tissues. This animal model, in which tissue perfusion and cardiac contractility are improved and muscle patch degradation is hindered, might find application in humans suffering from ischemic heart failure. For example, cardiomyoplasty with a latissimus dorsi muscle that has been coated with autologous BMCs might be used to enhance cardiac regeneration. Before clinical trials of our approach can be attempted, however, other experiments aimed at elucidating the mechanism(s) of action at work in our murine model are warranted.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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