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Effects of cardiac patches engineered with bone marrow-derived mononuclear cells and PGCL scaffolds in a rat myocardial infarction model
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Biomaterials 28 (2007) 641–649 www.elsevier.com/locate/biomaterials
Effects of cardiac patches engineered with bone marrow-derived mononuclear cells and PGCL scaffolds in a rat myocardial infarction model Hainan Piaoa,1, Jin-Sook Kwona,1, Shuguang Piaoa, Ju-Hee Sohna, Yeong-Shin Leea, Jang-Whan Baea, Kyung-Kuk Hwanga, Dong-Woon Kima, Oju Jeonb, Byung-Soo Kimc, Young-Bae Parkd,2, Myeong-Chan Choa,,2 a
Department of Cardiology, College of Medicine, Chungbuk National University, 62 Gaesin-dong, Heungdeok-gu, Cheongju 361-763, Korea b Department of Chemical Engineering, Hanyang University, 17 Haengdang 1-dong, Seongdong-gu, Seoul 133-791, Korea c Department of Bioengineering, Hanyang University, 17 Haengdang 1-dong, Seongdong-gu, Seoul 133-791, Korea d Department of Cardiology, College of Medicine, Seoul National University, Yeongeon-dong, Jongno-gu, Seoul 110-744, Korea Received 5 July 2006; accepted 8 September 2006 Available online 10 October 2006
Abstract Little is known about the cardioprotective effects against heart failure (HF), the effects on differentiation of bone marrow-derived mononuclear cell (BMMNC), and the biocompatibility of BMMNC-seeded biodegradable poly-glycolide-co-caprolactone (PGCL) scaffolds in a myocardial infarction (MI) animal model. This study hypothesized that implantation of a BMMNC-seeded PGCL scaffold into the epicardial surface in a rat MI model would be biocompatible, induce BMMNC migration into infarcted myocardium, and effectively improve left ventricular (LV) systolic dysfunction. One week after the implantation of a BMMNC-seeded PGCL scaffold, BMMMC showed migration into the epicardial region. Four weeks after implantation, augmented neovascularization was observed in infarcted areas and in infarct border zones. Some BMMNCs exhibited the presence of a-MHC and troponin I, markers of differentiation into cardiomyocytes. In echocardiographic examinations, BMMNC-seeded PGCL scaffold and non-cell-seeded simple PGCL scaffold groups effectively reduced progressive LV dilatation and preserved LV systolic function as compared to control rat MI groups. Thus, BMMNC-seeded PGCL scaffolding influences BMMNC migration, differentiation to cardiomyocytes, and induction of neovascularization, ultimately effectively lessening LV remodeling and progressive LV systolic dysfunction. PGCL scaffolding can be considered as an effective treatment alternative in MI-induced advanced HF. r 2006 Elsevier Ltd. All rights reserved. Keywords: Myocardial infarction; Heart failure; Stem cell; Polymer
1. Introduction Heart failure (HF) is a major cause of death in developed countries. An important cause of HF is myocardial infarction (MI). MI provokes massive loss of contractile Corresponding author. Tel.: +82 43 269 6356; fax: +82 43 273 3252.
E-mail addresses: [email protected] (Y.-B. Park), [email protected] (M.-C. Cho). 1 Hainan Piao and Jin-Sook Kwon also equally contributed to this article as co-first authors. 2 Young-Bae Park and Myeong-Chan Cho equally dedicated to this article as co-corresponding authors. 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.09.009
functioning cardiomyocytes, and infarcted lesions are replaced by non-functioning fibrotic tissue. Myocardial regenerative capability against ischemic cell loss is very limited, and ventricular remodeling and progressive ventricular dysfunction are eminent. Heart transplantation is ultimately the only treatment for end-stage HF. Efforts to regenerate functional myocardial tissue were initially pursued through cell grafting by syringe injection directly into the ventricular wall or the coronary vessels [1]. Proof of principle for cardiac cell implantation in the heart was first achieved nearly 10 years ago using genetically selected embryonic stem cell-derived cardiomyocytes [1–3].
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Many researchers have demonstrated that implantation of fetal or neonatal cardiomyocytes, smooth muscle cells, skeletal myoblasts, or bone marrow-derived cells into sites of MI could induce effective angiogenesis and/or improve left ventricular (LV) systolic function [4–9]. Autologous bone marrow-derived cells are especially attractive in the treatment of MI in the clinical field because these cells can be differentiated into cardiomyocytes, endothelial cells, or both [10–13] without significant immunologic rejection. Previous data indicate that the cell therapy is a promising treatment, but cell transfer efficiency and survival rate in recipient myocardium is very limited. In addition, simple cell supply via coronary artery or direct myocardial injection has proved deficient in preventing progressive LV dilation, due to lack of a mechanical barrier. Against this background, in order to augment cell transfer efficiency or survival and to build some physiologic barrier for preventing LV remodeling, stem cell-seeded biocompatible cardiac patches could be considered as a new therapeutic method in myoangiogenesis for infarctionrelated advanced HF. Already, in the case of large infarctions, attempts have been made to replace infarcted cardiac tissue with tissue-engineered cardiac patches made of biocompatible and bioabsorbable materials at a rate compatible with the repair process [14]. Threedimensional cardiac patches have been engineered using gelatin mesh, collagen gel, alginate, e-caprolactone-co-Llactide sponges reinforced with knitted poly-L-lactide fabric, and polyglycolic acid [10,15–18]. The ideal material for cardiac patches should be sufficiently strong to withstand the force of repeated contraction in the myocardium [19]. Poly[glycolide-co-caprolactone] (PGCL) scaffold is fairly elastic, suggesting that it could be employed to engineer a patch for mechanically dynamic environments such as the heart [20]. Beyond possessing the necessary mechanical elastic property, a good candidate scaffold should allow proper cellular interactions, such as the transferring of mechanical signals to the seeded cells. PGCL scaffolds both possess good elastic mechanical properties and can promote appropriate cellular interaction [10]. In the present study, we investigated whether tissueengineered cardiac patches incorporating PGCL scaffolds laden with bone marrow-derived mononuclear cells (BMMNCs) in rat MI model could prevent cardiac remodeling and improve LV systolic dysfunction. Rat BMMNCs were seeded onto PGCL scaffolds and implanted on the epicardial surface over infracted areas and adjacent normal myocardium. Four weeks after implantation, LV function was examined with echocardiography and left ventricular end-diastolic pressure (LVEDP) analysis by catheterization, and tissue regeneration and neovascularization were analyzed by histological and immunohistochemical staining. To our knowledge, this report is the first in the literature addressing implantation of BMMNC-seeded cardiac scaffolds for regeneration of the myocardium.
2. Materials and methods 2.1. Scaffold characterization PGCL scaffold was obtained courtesy of Dr. B.S. Kim (Department of Chemical Engineering, Hanyang University, Seoul, Korea). Appropriate biodegradable and biocompatible properties of PGCL scaffolds were already well defined in previous literature [10,20].
2.2. BMMNC culture and cell seeding onto PGCL scaffold Bone marrow was flushed from the femurs and tibias of 200–220 g Sprague-Dawley rats (Samtaco, Kyunggi-do, Korea) into Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Gaithersburg, MD). The cell suspension was loaded on a Histopaque density gradient (specific gravity ¼ 1.077, Sigma, St. Louis, MO) and centrifuged for 20 min at 230g. BMMNCs were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco BRL) and 100 unit/ml penicillin and streptomycin (Gibco BRL) in humidified air with 5% CO2 at 37 1C. The culture medium was changed every 2 days. Cultured BMMNCs were labeled with 100 ng/ml of 40 ,60 -diamidino-2-phenylindole hydrochloride (DAPI, Sigma) at 37 1C for 30 min, after which the labeled cells were washed three times with PBS. BMMNCs (2 106 cells) were uniformly seeded onto PGCL scaffolds (5 mm 5 mm). The seeded scaffolds were maintained in vitro in DMEM supplemented with 10% (v/v) FBS and 100 unit/ml penicillin and streptomycin in humidified air with 5% CO2 at 37 1C for 2 days prior to implantation. Non-cell-seeded PGCL scaffold was prepared using the same method but without BMMNC.
2.3. Scanning electron microscopy Patches were plated and cultured for 2 days and were fixed in 1% (v/v) buffered glutaraldehyde and 0.1% (v/v) buffered formaldehyde for 30 min and 24 h, respectively, dehydrated with a graded ethanol series, and dried. The dried samples were mounted on aluminum stubs and sputter-coated with gold. A scanning electron microscope (SEM) (JSM-6330F, JEOL, Tokyo, Japan) was used to image the samples.
2.4. Rat MI model and scaffold implantation Sprague-Dawley rats weighing 200–230 g were anesthetized with ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (2.5 mg/kg). The anesthetized rats were ventilated under positive pressure using a rodent ventilator (Model 683, Harvard Apparatus, South Natick, MA). A left thoracotomy was performed on each rat in the fourth intercostal space and the pericardium was opened. The left coronary artery was enclosed and occluded within the myocardium between the left atrial appendage and right ventricular outflow tract, using a curved needle and 6-0 silk, and the chest was then closed in layers and the pneumothorax was evacuated. After 5 h, the suture which occluded the left coronary artery was removed. All care and handling of animals were performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH publication 85-23, revised 1985). Seven days after generation of MI, rats were randomized into three treatment groups, which received implantation of BMMNCseeded scaffold, implantation of non-cell-seeded simple scaffold, or sham operation (control group). BMMNC-seeded or non-cellseeded PGCL scaffolds were simultaneously sutured with 7-0 silk onto the epicardial surface over regions of infarcted myocardium and adjacent infarction border zones. Four sutures into the infarct and border zones were simultaneously performed for the sham-operated control group.
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2.5. Histological and immunohistochemical examinations Hearts were excised and transversely sectioned across the infarct territory into two blocks. The tissue blocks were frozen in liquid nitrogen with Tissue-Tek OCT compound (Miles Laboratories, Naperville, IL) and stored at 70 1C. Specimens from the implantation sites were stained with hematoxylin and eosin (H&E). Fibrotic tissue was analyzed by the Masson-trichrome method. For immunohistochemical analyses, 4 mmthick sections were stained using antibodies against cardiac myosin heavy chain (Santa Cruz, Santa Cruz, CA), troponin I (Santa Cruz), and von Willebrand factor (vWF, Sigma-Aldrich). For detection of myosin heavy chain and troponin I, Texas Red-labeled anti-mouse IgG or anti-goat IgG secondary antibodies were used. The above markers were used for detecting neoangiogenesis and differentiation of BMMNC into cardiomyocytes.
2.6. Functional evaluation of infarcted myocardium Transthoracic echocardiography was performed at 7 days after MI, before the implantation of scaffold (to obtain a baseline echocardiogram), and 4 weeks after implantation. Rats were anesthetized with 50 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride for echocardiographic examination. A commercial echocardiographic machine (128-XP, Acuson, Mountain View, CA) with a 15 MHz linear transducer was used for the evaluation of LV dimension and systolic function. Under the left lateral decubitus position, M-mode tracing of the LV was obtained close to the papillary muscle level by using the parameter short axis imaging plane. All dimension and thickness data for LV were measured using the American Society of Echocardiography leading-edge technique, and the results are presented as the average of three measurements. The fractional shortening (FS) of LV was expressed as FS (%) ¼ [(LVIDd, LV internal dimension on end diastoleLVIDs or LV internal dimension on end systole)/(LVIDd)] 100. All measurements were averaged for three consecutive cardiac cycles and were made by an experienced technician who was blinded to the treatment group. Following echocardiography, hemodynamic evaluation was performed in closed-chest rats as described previously [18,21]. After general anesthesia, an arterial catheter (PE 50 tube, Clay Adams, NJ) was advanced into the LV through the right carotid artery. The pressure curve of the LV was recorded and monitored continuously. Pressure variables were averaged for three consecutive cardiac cycles and were made by an experienced technician who blinded to the treatment group. Exclusively, LVEDP was recorded and statistically analyzed.
2.7. Statistical analysis Data were obtained from three animals per group and expressed as the mean7standard deviation. Statistical analysis was performed using the
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unpaired Student’s t-test. A value of po0:05 was considered to be statistically significant.
3. Results 3.1. Characteristics of BMMNC-seeded scaffold The seeded BMMNC adhered well to the PGCL scaffolds. SEM examination and histological analysis showed that a high density of BMMNC was present on the PGCL scaffolds (Fig. 1). 3.2. Microscopic and histological analysis Prior to the main experiment, BMMNC-seeded PGCL scaffolds were implanted on the epicardial surface of normal heart in order to evaluate the safety of the PGCL scaffold. At 1 week, the BMMNC-seeded PGCL scaffold was well attached to cardiac muscle and the seeded cells migrated into epicardial region effectively (data not shown). Following the safety evaluation, BMMNC-seeded PGCL scaffolds were implanted on the epicardial surface over infarcted hearts. By visual detection at 4 weeks after implantation, scaffolds were covered by a thin translucent connective tissue that was enriched with newly constituted blood vessels. This extensive network of newly constituted vessels in the scaffold emerged from the neighboring coronary network. Similar results were obtained in the non-cell-seeded scaffold implantation group also. However, the density of new vessel development was higher in the BMMNC-seeded patch group than in the non-cellseeded patch group. Addition to this finding, implanted PGCL scaffold limited infarct region expansion. Control group showed marked ventricular dilatation compared to PGCL scaffold implanted heart (Fig. 2). In the gross specimens which were stained with Masson’s trichrome, BMMNC-seeded and non-cell-seeded PGCL scaffold groups reduced fibrosis tissue area and attenuated LV remodeling effectively compared to sham-operated control group at 2 weeks after implantation (Fig. 3). Histological examination of thin sections of the scaffold identified increased vascular formation. Well-organized vessels were
Fig. 1. (A) BMMNCs (red) attach well to the PGCL scaffold (white amorphous area) as shown by H&E staining ( 400). (B) The PGCL scaffold was seen to be covered with abundant numbers of BMMNC in SEM ( 1100).
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Fig. 2. Only four simple sutures were performed in the MI area in the control (A). Non-cell-seeded PGCL scaffolds (B) and BMMNC-seeded PGCL scaffolds were implanted for study groups. Translucent connective tissue containing new blood vessels emerged from the adjacent coronary network in (B) and (C). This network did not exist in the control group (A), and the highest density was obtained in the BMMNC-seeded PGCL scaffold group.
Fig. 3. Sham-operated control heart shows marked wall thinning in the infarcted area (A). Reduced fibrotic area and preserved cardiac muscle areas are observed in non-cell-seeded (B) and BMMNC-seeded PGCL scaffold (C) group hearts in Masson’s trichrome staining.
Fig. 4. Control group heart (A) shows massive fibrosis in infarcted area. However, abundant new organized vessels are observed in both non-cell-seeded PGCL scaffold (B) and BMMNC-seeded PGCL scaffold (C) groups. Cardiac muscles are less damaged in the BMMNC-seeded PGCL scaffold group (C).
observed in the boundary region between scaffold and epicardial region in BMMNC-seeded PGCL scaffolds implantation group but this neovascularization was not founded at non-cell-seeded simple scaffold implantation group and sham-operated control group (Fig. 4). Even though we had not done immunosuppressive therapy before or after scaffold implantation to infarcted rat heart, there was no evidence of marked inflammatory cell infiltration or immunologic rejection in cardiac tissue (Fig. 4). Scattered numerous bright-blue dot shaped implanted BMMNCs were detected in the amouphous bright-blue PGCL scaffold surface under fluorescence microscopic examination (Fig. 5A). BMMNCs survived well in the PGCL scaffold which was implanted on the epicardial surface of infarcted and its adjacent normal myocardial tissue, and these cells penetrated well into
recipient myocardium through epicardium (Fig. 5B and C). These penetrated BMMNCs showed cardiomyocyte phenotypes under the staining for myosin heavy chain and troponin I. With this immunohistochemical analysis, we have known that some portion of BMMNCs differentiated into cardiomyocytes in the infarcted rat hearts (Fig. 6). In addition, implanted PGCL scaffolds limited expansion of the infarct region. The sham-operated control hearts showed marked ventricular dilatation compared to PGCL scaffold-implanted hearts (Fig. 2). In the gross specimens which were stained with Masson’s trichrome, BMMNCseeded and non-cell-seeded PGCL scaffold groups exhibited both reduced fibrotic tissue areas and effectively attenuated LV remodeling compared to the sham-operated control group at 2 weeks after implantation (Fig. 3). Histological examination of thin sections of the scaffold
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Fig. 5. (A) Immuhistochemical staining for DAPI-positive BMMNC (blue dots) in PGCL scaffold (white-gray amorphous material) which was implanted in infarcted and adjacent normal myocardium. BMMNCs survive effectively in the scaffold, and some of these penetrate into the recipient myocardium (C) via the epicardial area (B).
Fig. 6. Some DAPI-labeled BMMNC show a cardiomyocyte phenotype in MHC, as well as TnI immunochemical staining at 4 weeks after transplantation with PGCL scaffold to infarcted heart ( 1200). (A, D) DAPI-positive implanted BMMNC. (B) Red-labeled MHC. (C) Merged figure of (A) and (B). (E) Red-labeled TnI. (F) Merged figure of (D) and (E).
revealed increased vascular formation. Well-organized vessels were observed in the boundary region between the scaffold and epicardium in the BMMNC-seeded PGCL scaffold implantation group, but this neovascularization
was not found in the non-cell-seeded scaffold implantation group or the sham-operated control group (Fig. 4). Even though we had not performed immunosuppressive therapy before or after scaffold implantation into infarcted rat
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hearts, there was no evidence of marked inflammatory cell infiltration or immunologic rejection in cardiac tissue (Fig. 4). Under fluorescence microscopic examination, numerous scattered implanted BMMNCs were detected as bright-blue dots against the amorphous bright-blue background of the PGCL scaffold surface (Fig. 5A).
BMMNCs survived well in the PGCL scaffolds which were implanted on the epicardial surface of infarcted and adjacent normal myocardial tissue, and these cells penetrated well into recipient myocardium through the epicardium (Fig. 5B and C). These penetrated BMMNCs exhibited cardiomyocyte phenotypes, as indicated by staining for myosin heavy chain and troponin I. By this immunohistochemical analysis, we determined that a portion of the BMMNC differentiated into cardiomyocytes in the infarcted rat hearts (Fig. 6).
3.3. Echocardiographic study and intracardiac pressure monitoring
Fig. 7. Echocardiographic examination at 4 weeks after myocardial infarction. Control group heart (A) shows LV dilation and significant LV wall thinning. Non-cell-seeded PGCL scaffold group heart (B) and BMMNC-seeded PGCL scaffold group heart (C) show limited LV remodeling and preserved anterior wall thickness.
To evaluate the influence of the scaffold on LV remodeling and function, a series of echocardiographic examinations were conducted. After coronary artery ligation, hearts of the sham-operated control group developed a typical course of LV remodeling and HF that complicated anterior wall MI (Fig. 7). BMMNC-seeded PGCL scaffolds and non-cell-seeded PGCL scaffolds groups, in contrast, showed attenuation of LV dilation and LV dysfunction compared to the sham-operated group. The end diastolic LV dimensions in rats treated with BMMNC-seeded PGCL scaffolds (8.670.6 mm) and non-cell-seeded simple PGCL scaffolds (8.770.8 mm) were significantly smaller than in the sham-operated group (11.170.5 mm). The end systolic LV dimensions of rats implanted with BMMNC-seeded PGCL scaffolds (5.870.9 mm) and non-cell-seeded PGCL scaffolds (6.07 1.6 mm) were also smaller than in the sham-operated group (9.470.4 mm). Consistent with this attenuation in LV remodeling, FS in both the BMMNC-seeded PGCL scaffold group and the non-cell-seeded PGCL scaffold group was higher than in the sham-operated group (32.578.6% and 31.8711.9% vs. 15.674.3%). LVEDP was significantly lower in the BMMNC-seeded scaffold group (2.572.3 mmHg) and the non-cell-seeded PGCL scaffold group (2.370.6 mmHg) than in the sham-operated group (11.473.8 mmHg) (Table 1).
Table 1 Echocardiographic and LV hemodynamic parameters at 4 weeks after PGCL scaffold implantations Control group Echocardiographic examination LVIDd (mm) 11.170.5 LVIDs (mm) 9.470.4 FS (%) 15.674.3 LVPW thickness 1.770.1 IVS thickness 1.370.11 Hemodynamic parameters LVEDP (mmHg) 11.473.8
Non-cell-seeded PGCL scaffold group
BMMNC-seeded PGCL scaffold group
8.770.8y 6.071.6* 31.8711.9* 1.970.2 1.770.2y
8.670.6y 5.870.9y 32.578.6y 1.870.2 1.570.1*
2.370.6*
2.572.3y
BMMNC, bone marrow mononuclear cell; PGCL, poly-glycolide-co-caprolactone; LVIDd, left ventricular internal dimension at diastole; LVIDds, left ventricular internal dimension at systole; FS, fractional shortening; LVEDP, left ventricular end-diastolic pressure. *po0.05 compared to control group. y po0.01 compared to control group.
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4. Discussion For patients with an extensive MI, surgical remodeling procedures such as the Dor operation or endoventricular patch procedure (EVCPP) can improve LV systolic function [19–22]. However, even after successful repair with these procedures, some patients suffer recurrent LV remodeling [19–26]. Mitral regurgitation has also been reported as a late complication of these procedures in association with recurrent dilatation [23–26]. Nishina et al. reported that improvements in LV size and function after plication in a rat ischemic cardiomyopathic model was transient because of residual scar remodeling and changes in LV geometry [23]. In a sheep model of MI, suturing a polyproplene mesh over the infarcted area prevented progressive LV expansion. This technique maintained normal ventricular geometry and heart function for 8 weeks despite substantial tissue losses [25]. Similarly, Leor et al. showed that implantation of a bioengineered fetal rat cardiomyocyte graft onto the surface of a MI attenuated ventricular dilatation [27]. The beneficial effects of this technique may be explained by the elastic properties of the bioengineered vascular smooth muscle graft. Like this bioengineered vascular smooth muscle graft, it is well known that PGCL scaffold has appropriate biodegradability and biocompatibility [26]. In the present study, we investigated the effects of tissueengineered cardiac patches consisting of BMMNC and PGCL scaffolds on attenuation of LV dysfunction and on induction of neovascularization in a rat model of infarcted heart. Rat BMMNCs were seeded onto PGCL scaffolds and implanted onto the epicardial surface over infarcted regions and adjacent normal heart. Four weeks after implantation, BMMNCs survived well in the scaffold, integrated into the host myocardium, and induced tissue preservation and neovascularization. Both patch groups had significantly improved LV function compared to control group. These favorable results can be explained as consequences of the cardioprotective effects induced by bioengineered scaffold and applied stem cells. PGCL scaffold alone, acting as a mechanical barrier, was able to prevent accelerated LV dilation after MI, and implanted BMMNC produced positive effects on both neovascularization and cardiomyocyte regeneration in ischemically jeopardized myocardium. With BMMNC, tissue ischemia was relieved and contractile dysfunction was attenuated by neovascularization. Matsubayashi et al. reported that repairing LV aneurysms with patches for a modified EVCPP composed of vascular smooth muscle cell-laden synthetic PCLA (sponge polymer of e-caprolactone-coL-lactide reinforced with knitted poly-L-lactide fabric) attenuated LV volume and systolic dysfunction in a rat MI model [28]. In this study, only the cell-seeded synthetic patches showed cardioprotective effects. This disparity with the previous result may be due to differences in the cell types applied, scaffold implantation time points after
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coronary artery ligation, and/or patch implantation methods. The type of cells chosen for implantation in treatment of MI is a vitally important question. Autologous cell implantation is intriguing in that it can avoid the problem of immunorejection. Obtaining autologous cells from bone marrow is attractive because marrow is easily obtained clinically and contains endothelial progenitor cells that could contribute to new blood vessel formation [28]. In addition, implantation of bone marrow-derived cells could induce regeneration of cardiac muscle and blood vessels in infarcted cardiac tissue, which is critical for recovery of heart function [31]. In clinical trials, intracoronary transplantation of bone marrow-derived cells into infarcted myocardium resulted in myocardial regeneration and neovascularization, and transendocardial transplantation of bone marrow-derived cells into infarcted myocardium safely promoted neovascularization and improved LV function [29–31]. In view of these past favorable results with BMMNC, we chose them for this study. A portion of the implanted BMMNCs seeded on PGCL scaffolds exhibited cardiomyocyte markers in this study. In previous reports, bone marrow stem cell transplanted into infarcted heart differentiated into smooth muscle cells, cardiomyocytes, and endothelial cells. Neovascularization was significantly greater in our BMMNC-seeded PGLC scaffold group than in the non-cell-seeded scaffold or sham-operated control groups. Neovascularization contributed to prolonged cell survival in the patches, and this may lead to improved collateral flow and augmented cardiomyocyte contractility and thus result in improvement of LV function. Even after the biodegradation of PGCL scaffold, this neovascularization in the ischemic zone may contribute to protection of the tissue against MI and to attenuation of subsequent progressive LV remodeling and LV systolic dysfunction. Implantation of organically structured stem cells on a biomaterial patch offers several advantages over direct injection of dissociated cells into infarcted cardiac tissue. In case of direct injection of dissociated cells, it is difficult to control shape and location in cardiac tissue [32], but with implantation of cells seeded on scaffolds, the polymeric scaffold provides a temporary support for the seeded cells until they produce their own extracellular matrix. This newly formed extracellular matrix around the cardiac patch maintains the structure and elasticity of the ventricular wall. Among biodegradable polymer scaffolds, PGCL scaffold is especially attractive for use in cardiac patches. A cardiac patch implanted onto the epicardial surface is under continuous contraction, so it needs to be sufficiently strong and elastic to resist damage from the contracting myocardium. The PGCL scaffolds used in this study were found to be highly elastic and flexible in mechanical tests, and they proved to be suitable in the mechanically dynamic environment of the heart. The combined advantages of PGCL scaffolds and BMMNC, with their vasculocardiomyogenic properties, were able to
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attenuate progressive LV dysfunction associated with MI in the rat model used in this study. 5. Conclusion BMMNC-seeded PGCL scaffold implantation effectively attenuated LV remodeling and LV systolic dysfunction in a rat myocardial infarction (MI) model. PGCL scaffold served as a mechanical barrier against progressive LV dilation in infarcted areas, and seeded BMMNCs survived, integrated into the myocardium, and effectively induced neovascularization. A portion of the implanted stem cells differentiated into cardiomyocytes. By this mechanism, BMMNC-seeded PGCL scaffold supplied contractile functioning cardiomyocytes, effectively reduced the ischemic area, and attenuated LV dilation, ultimately improving LV systolic function. The stem cell-seeded biocompatible scaffold can be applied as an alternative therapeutic modality for medically intractable advanced heart failure, especially for failure associated with MI. Acknowledgments This research was supported by a grant SC13121 and SC3150 [Dr. YB Park] from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea. References [1] Zammaretti P, Jaconi M. Cardiac tissue engineering: regeneration of the wounded heart. Curr Opin Biotechnol 2004;15:430–4. [2] Klug MJ, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embryonic stem cells from stable intracardiac grafts. J Clin Invest 1996;98:216–24. [3] Koh GY, Soonpaa MH, Klug MG, Pride HP, Copper BJ, Zipes DP, et al. Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs. J Clin Invest 1995;96:2034–42. [4] Li RK, Jia ZQ, Weisel RD, Mickle DA, Zhang J, Mohabeer MK, et al. Cardiomyocyte transplantation improves heart function. Ann Thorac Surg 1996;62:654–60. [5] Li RK, Jia ZQ, Weisel RD, Merante F, Mickle DA. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol 1999;31:513–22. [6] Jain M, DerSimonian H, Brenner DA, Ngoy S, Teller P, Edge AS, et al. Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation 2001;103:1920–7. [7] Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7:430–6. [8] Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 2001;104:1046–52. [9] Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–5.
[10] Lee SH, Kim BS, Kim SH, Choi SW, Jeong SI, Kwon IK, et al. Elastic biodegradable poly(glycolide-co-caprolactone) scaffold for tissue engineering. J Biomed Mater Res 2003;66:29–37. [11] Tomita S, Li RK, Weisel RD, Mickle DA, Kim EJ, Sakai T, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100:II247–56. [12] Saito T, Dennis JE, Lennon DP, Young RG, Caplan AI. Myogenic expression of mesenchymal stem cells within myotubes of mdx mice in vitro and in vivo. Tissue Eng 1995;1:327–43. [13] Radisic M, Euloth M, Yang L, Langer R, Freed LE, VunjakNovakovic G. High-density seeding of myocyte cells for cardiac tissue engineering. Biotechnol Bioeng 2003;20:403–14. [14] Li RK, Jia ZQ, Weisel RD, Mickle DA, Choi A, Yau TM. Survival and function of bioengineered cardiac grafts. Circulation 1999; 100:II63–9. [15] Eschenhagen T, Fink C, Remmers U, Scholz H, Wattchow J, Weil J, et al. Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J 1997;11:683–94. [16] Dar A, Shachar M, Leor J, Cohen S. Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds. Biotechnol Bioeng 2002;8:305–12. [17] Ozawa T, Mickle DA, Weisel RD, Koyama N, Ozawa S, Li RK. Optimal biomaterial for creation of autologous cardiac grafts. Circulation 2002;106:I176–82. [18] Kim BS, Mooney DJ. Scaffolds for engineering smooth muscle under cyclic mechanical strain conditions. J Biomech Eng 2000; 122:210–5. [19] Di Donato M, Sabatier M, Dor V, Toso A, Maioli M, Fantini F. Akinetic versus by skinetic post infarction scar: relation to surgical outcome in patients undergoing endoventricular circular patch plasty repair. J Am Coll Cardiol 1997;29:1569–75. [20] Di Donato M, Sabatier M, Dor V, Genesini GF, Toso A, Maioli M, et al. Effects of the Dor procedure on left ventricular dimension and shape and geometric correlates of mitral regurgitation one year after surgery. J Thorac Cardiovasc Surg 2001;121:91–6. [21] Dor V, Sabatier M, Di Donato M, Montiglio F, Toso A, Maioli M. Efficacy of endoventricular patch plasty in large postinfarction akinetic scar and severe left ventricular dysfunction: comparison with a series of large dyskinetic scars. J Thorac Cardiovasc Surg 1998;116:50–9. [22] Sinatra R, Macrina F, Braccio M, Melina G, Luzi G, Ruvolo G, et al. Left ventricular aneurysmectomy; comparison between two techniques; early and late results. Eur J Cardiothorac Surg 1997; 12:291–7. [23] Nishina T, Nishimura K, Yuasa S, Miwa S, Nomota T, Sakakibara Y, et al. Initial effects of the left ventricular repair by plication may not last long in a rat ischemic cardiomyopathy model. Circulation 2001;104:I241–5. [24] Sakakibara Y, Tambara K, Lu F, Nishina T, Sakaguchi G, Nagaya N, et al. Combined procedure of surgical repair and cell transplantation for left ventricular aneurysm: an experimental study. Circulation 2002;106:I193–7. [25] Kelley ST, Malekan R, Gorman III JH, Jackson BM, Gorman RC, Suzuki Y, et al. Restraining infarct expansion preserves left ventricular geometry and function after acute anteroapical infarction. Circulation 1999;99:135–42. [26] Matsubayashi K, Fedak PW, Mickle DA, Weisel RD, Ozawa T, Li RK. Improved left ventricular aneurysm repair with bioengineered vascular smooth muscle grafts. Circulation 2003;108:II219–25. [27] Leor J, Aboulafia-Etzion S, Dar A. Bioengineered cardiac grafts: a new approach to repair the infarcted myocardium? Circulation 2000;102:III56–61. [28] Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 2002;109:337–46. [29] Eschenhagen T, Fink C, Remmers U, Scholz H, Wattchow J, Weil J, et al. Three-dimensional reconstitution of embryonic cardiomyocytes
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Biomaterials 28 (2007) 641–649 www.elsevier.com/locate/biomaterials
Effects of cardiac patches engineered with bone marrow-derived mononuclear cells and PGCL scaffolds in a rat myocardial infarction model Hainan Piaoa,1, Jin-Sook Kwona,1, Shuguang Piaoa, Ju-Hee Sohna, Yeong-Shin Leea, Jang-Whan Baea, Kyung-Kuk Hwanga, Dong-Woon Kima, Oju Jeonb, Byung-Soo Kimc, Young-Bae Parkd,2, Myeong-Chan Choa,,2 a
Department of Cardiology, College of Medicine, Chungbuk National University, 62 Gaesin-dong, Heungdeok-gu, Cheongju 361-763, Korea b Department of Chemical Engineering, Hanyang University, 17 Haengdang 1-dong, Seongdong-gu, Seoul 133-791, Korea c Department of Bioengineering, Hanyang University, 17 Haengdang 1-dong, Seongdong-gu, Seoul 133-791, Korea d Department of Cardiology, College of Medicine, Seoul National University, Yeongeon-dong, Jongno-gu, Seoul 110-744, Korea Received 5 July 2006; accepted 8 September 2006 Available online 10 October 2006
Abstract Little is known about the cardioprotective effects against heart failure (HF), the effects on differentiation of bone marrow-derived mononuclear cell (BMMNC), and the biocompatibility of BMMNC-seeded biodegradable poly-glycolide-co-caprolactone (PGCL) scaffolds in a myocardial infarction (MI) animal model. This study hypothesized that implantation of a BMMNC-seeded PGCL scaffold into the epicardial surface in a rat MI model would be biocompatible, induce BMMNC migration into infarcted myocardium, and effectively improve left ventricular (LV) systolic dysfunction. One week after the implantation of a BMMNC-seeded PGCL scaffold, BMMMC showed migration into the epicardial region. Four weeks after implantation, augmented neovascularization was observed in infarcted areas and in infarct border zones. Some BMMNCs exhibited the presence of a-MHC and troponin I, markers of differentiation into cardiomyocytes. In echocardiographic examinations, BMMNC-seeded PGCL scaffold and non-cell-seeded simple PGCL scaffold groups effectively reduced progressive LV dilatation and preserved LV systolic function as compared to control rat MI groups. Thus, BMMNC-seeded PGCL scaffolding influences BMMNC migration, differentiation to cardiomyocytes, and induction of neovascularization, ultimately effectively lessening LV remodeling and progressive LV systolic dysfunction. PGCL scaffolding can be considered as an effective treatment alternative in MI-induced advanced HF. r 2006 Elsevier Ltd. All rights reserved. Keywords: Myocardial infarction; Heart failure; Stem cell; Polymer
1. Introduction Heart failure (HF) is a major cause of death in developed countries. An important cause of HF is myocardial infarction (MI). MI provokes massive loss of contractile Corresponding author. Tel.: +82 43 269 6356; fax: +82 43 273 3252.
E-mail addresses: [email protected] (Y.-B. Park), [email protected] (M.-C. Cho). 1 Hainan Piao and Jin-Sook Kwon also equally contributed to this article as co-first authors. 2 Young-Bae Park and Myeong-Chan Cho equally dedicated to this article as co-corresponding authors. 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.09.009
functioning cardiomyocytes, and infarcted lesions are replaced by non-functioning fibrotic tissue. Myocardial regenerative capability against ischemic cell loss is very limited, and ventricular remodeling and progressive ventricular dysfunction are eminent. Heart transplantation is ultimately the only treatment for end-stage HF. Efforts to regenerate functional myocardial tissue were initially pursued through cell grafting by syringe injection directly into the ventricular wall or the coronary vessels [1]. Proof of principle for cardiac cell implantation in the heart was first achieved nearly 10 years ago using genetically selected embryonic stem cell-derived cardiomyocytes [1–3].
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Many researchers have demonstrated that implantation of fetal or neonatal cardiomyocytes, smooth muscle cells, skeletal myoblasts, or bone marrow-derived cells into sites of MI could induce effective angiogenesis and/or improve left ventricular (LV) systolic function [4–9]. Autologous bone marrow-derived cells are especially attractive in the treatment of MI in the clinical field because these cells can be differentiated into cardiomyocytes, endothelial cells, or both [10–13] without significant immunologic rejection. Previous data indicate that the cell therapy is a promising treatment, but cell transfer efficiency and survival rate in recipient myocardium is very limited. In addition, simple cell supply via coronary artery or direct myocardial injection has proved deficient in preventing progressive LV dilation, due to lack of a mechanical barrier. Against this background, in order to augment cell transfer efficiency or survival and to build some physiologic barrier for preventing LV remodeling, stem cell-seeded biocompatible cardiac patches could be considered as a new therapeutic method in myoangiogenesis for infarctionrelated advanced HF. Already, in the case of large infarctions, attempts have been made to replace infarcted cardiac tissue with tissue-engineered cardiac patches made of biocompatible and bioabsorbable materials at a rate compatible with the repair process [14]. Threedimensional cardiac patches have been engineered using gelatin mesh, collagen gel, alginate, e-caprolactone-co-Llactide sponges reinforced with knitted poly-L-lactide fabric, and polyglycolic acid [10,15–18]. The ideal material for cardiac patches should be sufficiently strong to withstand the force of repeated contraction in the myocardium [19]. Poly[glycolide-co-caprolactone] (PGCL) scaffold is fairly elastic, suggesting that it could be employed to engineer a patch for mechanically dynamic environments such as the heart [20]. Beyond possessing the necessary mechanical elastic property, a good candidate scaffold should allow proper cellular interactions, such as the transferring of mechanical signals to the seeded cells. PGCL scaffolds both possess good elastic mechanical properties and can promote appropriate cellular interaction [10]. In the present study, we investigated whether tissueengineered cardiac patches incorporating PGCL scaffolds laden with bone marrow-derived mononuclear cells (BMMNCs) in rat MI model could prevent cardiac remodeling and improve LV systolic dysfunction. Rat BMMNCs were seeded onto PGCL scaffolds and implanted on the epicardial surface over infracted areas and adjacent normal myocardium. Four weeks after implantation, LV function was examined with echocardiography and left ventricular end-diastolic pressure (LVEDP) analysis by catheterization, and tissue regeneration and neovascularization were analyzed by histological and immunohistochemical staining. To our knowledge, this report is the first in the literature addressing implantation of BMMNC-seeded cardiac scaffolds for regeneration of the myocardium.
2. Materials and methods 2.1. Scaffold characterization PGCL scaffold was obtained courtesy of Dr. B.S. Kim (Department of Chemical Engineering, Hanyang University, Seoul, Korea). Appropriate biodegradable and biocompatible properties of PGCL scaffolds were already well defined in previous literature [10,20].
2.2. BMMNC culture and cell seeding onto PGCL scaffold Bone marrow was flushed from the femurs and tibias of 200–220 g Sprague-Dawley rats (Samtaco, Kyunggi-do, Korea) into Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Gaithersburg, MD). The cell suspension was loaded on a Histopaque density gradient (specific gravity ¼ 1.077, Sigma, St. Louis, MO) and centrifuged for 20 min at 230g. BMMNCs were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco BRL) and 100 unit/ml penicillin and streptomycin (Gibco BRL) in humidified air with 5% CO2 at 37 1C. The culture medium was changed every 2 days. Cultured BMMNCs were labeled with 100 ng/ml of 40 ,60 -diamidino-2-phenylindole hydrochloride (DAPI, Sigma) at 37 1C for 30 min, after which the labeled cells were washed three times with PBS. BMMNCs (2 106 cells) were uniformly seeded onto PGCL scaffolds (5 mm 5 mm). The seeded scaffolds were maintained in vitro in DMEM supplemented with 10% (v/v) FBS and 100 unit/ml penicillin and streptomycin in humidified air with 5% CO2 at 37 1C for 2 days prior to implantation. Non-cell-seeded PGCL scaffold was prepared using the same method but without BMMNC.
2.3. Scanning electron microscopy Patches were plated and cultured for 2 days and were fixed in 1% (v/v) buffered glutaraldehyde and 0.1% (v/v) buffered formaldehyde for 30 min and 24 h, respectively, dehydrated with a graded ethanol series, and dried. The dried samples were mounted on aluminum stubs and sputter-coated with gold. A scanning electron microscope (SEM) (JSM-6330F, JEOL, Tokyo, Japan) was used to image the samples.
2.4. Rat MI model and scaffold implantation Sprague-Dawley rats weighing 200–230 g were anesthetized with ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (2.5 mg/kg). The anesthetized rats were ventilated under positive pressure using a rodent ventilator (Model 683, Harvard Apparatus, South Natick, MA). A left thoracotomy was performed on each rat in the fourth intercostal space and the pericardium was opened. The left coronary artery was enclosed and occluded within the myocardium between the left atrial appendage and right ventricular outflow tract, using a curved needle and 6-0 silk, and the chest was then closed in layers and the pneumothorax was evacuated. After 5 h, the suture which occluded the left coronary artery was removed. All care and handling of animals were performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH publication 85-23, revised 1985). Seven days after generation of MI, rats were randomized into three treatment groups, which received implantation of BMMNCseeded scaffold, implantation of non-cell-seeded simple scaffold, or sham operation (control group). BMMNC-seeded or non-cellseeded PGCL scaffolds were simultaneously sutured with 7-0 silk onto the epicardial surface over regions of infarcted myocardium and adjacent infarction border zones. Four sutures into the infarct and border zones were simultaneously performed for the sham-operated control group.
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2.5. Histological and immunohistochemical examinations Hearts were excised and transversely sectioned across the infarct territory into two blocks. The tissue blocks were frozen in liquid nitrogen with Tissue-Tek OCT compound (Miles Laboratories, Naperville, IL) and stored at 70 1C. Specimens from the implantation sites were stained with hematoxylin and eosin (H&E). Fibrotic tissue was analyzed by the Masson-trichrome method. For immunohistochemical analyses, 4 mmthick sections were stained using antibodies against cardiac myosin heavy chain (Santa Cruz, Santa Cruz, CA), troponin I (Santa Cruz), and von Willebrand factor (vWF, Sigma-Aldrich). For detection of myosin heavy chain and troponin I, Texas Red-labeled anti-mouse IgG or anti-goat IgG secondary antibodies were used. The above markers were used for detecting neoangiogenesis and differentiation of BMMNC into cardiomyocytes.
2.6. Functional evaluation of infarcted myocardium Transthoracic echocardiography was performed at 7 days after MI, before the implantation of scaffold (to obtain a baseline echocardiogram), and 4 weeks after implantation. Rats were anesthetized with 50 mg/kg ketamine hydrochloride and 10 mg/kg xylazine hydrochloride for echocardiographic examination. A commercial echocardiographic machine (128-XP, Acuson, Mountain View, CA) with a 15 MHz linear transducer was used for the evaluation of LV dimension and systolic function. Under the left lateral decubitus position, M-mode tracing of the LV was obtained close to the papillary muscle level by using the parameter short axis imaging plane. All dimension and thickness data for LV were measured using the American Society of Echocardiography leading-edge technique, and the results are presented as the average of three measurements. The fractional shortening (FS) of LV was expressed as FS (%) ¼ [(LVIDd, LV internal dimension on end diastoleLVIDs or LV internal dimension on end systole)/(LVIDd)] 100. All measurements were averaged for three consecutive cardiac cycles and were made by an experienced technician who was blinded to the treatment group. Following echocardiography, hemodynamic evaluation was performed in closed-chest rats as described previously [18,21]. After general anesthesia, an arterial catheter (PE 50 tube, Clay Adams, NJ) was advanced into the LV through the right carotid artery. The pressure curve of the LV was recorded and monitored continuously. Pressure variables were averaged for three consecutive cardiac cycles and were made by an experienced technician who blinded to the treatment group. Exclusively, LVEDP was recorded and statistically analyzed.
2.7. Statistical analysis Data were obtained from three animals per group and expressed as the mean7standard deviation. Statistical analysis was performed using the
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unpaired Student’s t-test. A value of po0:05 was considered to be statistically significant.
3. Results 3.1. Characteristics of BMMNC-seeded scaffold The seeded BMMNC adhered well to the PGCL scaffolds. SEM examination and histological analysis showed that a high density of BMMNC was present on the PGCL scaffolds (Fig. 1). 3.2. Microscopic and histological analysis Prior to the main experiment, BMMNC-seeded PGCL scaffolds were implanted on the epicardial surface of normal heart in order to evaluate the safety of the PGCL scaffold. At 1 week, the BMMNC-seeded PGCL scaffold was well attached to cardiac muscle and the seeded cells migrated into epicardial region effectively (data not shown). Following the safety evaluation, BMMNC-seeded PGCL scaffolds were implanted on the epicardial surface over infarcted hearts. By visual detection at 4 weeks after implantation, scaffolds were covered by a thin translucent connective tissue that was enriched with newly constituted blood vessels. This extensive network of newly constituted vessels in the scaffold emerged from the neighboring coronary network. Similar results were obtained in the non-cell-seeded scaffold implantation group also. However, the density of new vessel development was higher in the BMMNC-seeded patch group than in the non-cellseeded patch group. Addition to this finding, implanted PGCL scaffold limited infarct region expansion. Control group showed marked ventricular dilatation compared to PGCL scaffold implanted heart (Fig. 2). In the gross specimens which were stained with Masson’s trichrome, BMMNC-seeded and non-cell-seeded PGCL scaffold groups reduced fibrosis tissue area and attenuated LV remodeling effectively compared to sham-operated control group at 2 weeks after implantation (Fig. 3). Histological examination of thin sections of the scaffold identified increased vascular formation. Well-organized vessels were
Fig. 1. (A) BMMNCs (red) attach well to the PGCL scaffold (white amorphous area) as shown by H&E staining ( 400). (B) The PGCL scaffold was seen to be covered with abundant numbers of BMMNC in SEM ( 1100).
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Fig. 2. Only four simple sutures were performed in the MI area in the control (A). Non-cell-seeded PGCL scaffolds (B) and BMMNC-seeded PGCL scaffolds were implanted for study groups. Translucent connective tissue containing new blood vessels emerged from the adjacent coronary network in (B) and (C). This network did not exist in the control group (A), and the highest density was obtained in the BMMNC-seeded PGCL scaffold group.
Fig. 3. Sham-operated control heart shows marked wall thinning in the infarcted area (A). Reduced fibrotic area and preserved cardiac muscle areas are observed in non-cell-seeded (B) and BMMNC-seeded PGCL scaffold (C) group hearts in Masson’s trichrome staining.
Fig. 4. Control group heart (A) shows massive fibrosis in infarcted area. However, abundant new organized vessels are observed in both non-cell-seeded PGCL scaffold (B) and BMMNC-seeded PGCL scaffold (C) groups. Cardiac muscles are less damaged in the BMMNC-seeded PGCL scaffold group (C).
observed in the boundary region between scaffold and epicardial region in BMMNC-seeded PGCL scaffolds implantation group but this neovascularization was not founded at non-cell-seeded simple scaffold implantation group and sham-operated control group (Fig. 4). Even though we had not done immunosuppressive therapy before or after scaffold implantation to infarcted rat heart, there was no evidence of marked inflammatory cell infiltration or immunologic rejection in cardiac tissue (Fig. 4). Scattered numerous bright-blue dot shaped implanted BMMNCs were detected in the amouphous bright-blue PGCL scaffold surface under fluorescence microscopic examination (Fig. 5A). BMMNCs survived well in the PGCL scaffold which was implanted on the epicardial surface of infarcted and its adjacent normal myocardial tissue, and these cells penetrated well into
recipient myocardium through epicardium (Fig. 5B and C). These penetrated BMMNCs showed cardiomyocyte phenotypes under the staining for myosin heavy chain and troponin I. With this immunohistochemical analysis, we have known that some portion of BMMNCs differentiated into cardiomyocytes in the infarcted rat hearts (Fig. 6). In addition, implanted PGCL scaffolds limited expansion of the infarct region. The sham-operated control hearts showed marked ventricular dilatation compared to PGCL scaffold-implanted hearts (Fig. 2). In the gross specimens which were stained with Masson’s trichrome, BMMNCseeded and non-cell-seeded PGCL scaffold groups exhibited both reduced fibrotic tissue areas and effectively attenuated LV remodeling compared to the sham-operated control group at 2 weeks after implantation (Fig. 3). Histological examination of thin sections of the scaffold
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Fig. 5. (A) Immuhistochemical staining for DAPI-positive BMMNC (blue dots) in PGCL scaffold (white-gray amorphous material) which was implanted in infarcted and adjacent normal myocardium. BMMNCs survive effectively in the scaffold, and some of these penetrate into the recipient myocardium (C) via the epicardial area (B).
Fig. 6. Some DAPI-labeled BMMNC show a cardiomyocyte phenotype in MHC, as well as TnI immunochemical staining at 4 weeks after transplantation with PGCL scaffold to infarcted heart ( 1200). (A, D) DAPI-positive implanted BMMNC. (B) Red-labeled MHC. (C) Merged figure of (A) and (B). (E) Red-labeled TnI. (F) Merged figure of (D) and (E).
revealed increased vascular formation. Well-organized vessels were observed in the boundary region between the scaffold and epicardium in the BMMNC-seeded PGCL scaffold implantation group, but this neovascularization
was not found in the non-cell-seeded scaffold implantation group or the sham-operated control group (Fig. 4). Even though we had not performed immunosuppressive therapy before or after scaffold implantation into infarcted rat
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hearts, there was no evidence of marked inflammatory cell infiltration or immunologic rejection in cardiac tissue (Fig. 4). Under fluorescence microscopic examination, numerous scattered implanted BMMNCs were detected as bright-blue dots against the amorphous bright-blue background of the PGCL scaffold surface (Fig. 5A).
BMMNCs survived well in the PGCL scaffolds which were implanted on the epicardial surface of infarcted and adjacent normal myocardial tissue, and these cells penetrated well into recipient myocardium through the epicardium (Fig. 5B and C). These penetrated BMMNCs exhibited cardiomyocyte phenotypes, as indicated by staining for myosin heavy chain and troponin I. By this immunohistochemical analysis, we determined that a portion of the BMMNC differentiated into cardiomyocytes in the infarcted rat hearts (Fig. 6).
3.3. Echocardiographic study and intracardiac pressure monitoring
Fig. 7. Echocardiographic examination at 4 weeks after myocardial infarction. Control group heart (A) shows LV dilation and significant LV wall thinning. Non-cell-seeded PGCL scaffold group heart (B) and BMMNC-seeded PGCL scaffold group heart (C) show limited LV remodeling and preserved anterior wall thickness.
To evaluate the influence of the scaffold on LV remodeling and function, a series of echocardiographic examinations were conducted. After coronary artery ligation, hearts of the sham-operated control group developed a typical course of LV remodeling and HF that complicated anterior wall MI (Fig. 7). BMMNC-seeded PGCL scaffolds and non-cell-seeded PGCL scaffolds groups, in contrast, showed attenuation of LV dilation and LV dysfunction compared to the sham-operated group. The end diastolic LV dimensions in rats treated with BMMNC-seeded PGCL scaffolds (8.670.6 mm) and non-cell-seeded simple PGCL scaffolds (8.770.8 mm) were significantly smaller than in the sham-operated group (11.170.5 mm). The end systolic LV dimensions of rats implanted with BMMNC-seeded PGCL scaffolds (5.870.9 mm) and non-cell-seeded PGCL scaffolds (6.07 1.6 mm) were also smaller than in the sham-operated group (9.470.4 mm). Consistent with this attenuation in LV remodeling, FS in both the BMMNC-seeded PGCL scaffold group and the non-cell-seeded PGCL scaffold group was higher than in the sham-operated group (32.578.6% and 31.8711.9% vs. 15.674.3%). LVEDP was significantly lower in the BMMNC-seeded scaffold group (2.572.3 mmHg) and the non-cell-seeded PGCL scaffold group (2.370.6 mmHg) than in the sham-operated group (11.473.8 mmHg) (Table 1).
Table 1 Echocardiographic and LV hemodynamic parameters at 4 weeks after PGCL scaffold implantations Control group Echocardiographic examination LVIDd (mm) 11.170.5 LVIDs (mm) 9.470.4 FS (%) 15.674.3 LVPW thickness 1.770.1 IVS thickness 1.370.11 Hemodynamic parameters LVEDP (mmHg) 11.473.8
Non-cell-seeded PGCL scaffold group
BMMNC-seeded PGCL scaffold group
8.770.8y 6.071.6* 31.8711.9* 1.970.2 1.770.2y
8.670.6y 5.870.9y 32.578.6y 1.870.2 1.570.1*
2.370.6*
2.572.3y
BMMNC, bone marrow mononuclear cell; PGCL, poly-glycolide-co-caprolactone; LVIDd, left ventricular internal dimension at diastole; LVIDds, left ventricular internal dimension at systole; FS, fractional shortening; LVEDP, left ventricular end-diastolic pressure. *po0.05 compared to control group. y po0.01 compared to control group.
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4. Discussion For patients with an extensive MI, surgical remodeling procedures such as the Dor operation or endoventricular patch procedure (EVCPP) can improve LV systolic function [19–22]. However, even after successful repair with these procedures, some patients suffer recurrent LV remodeling [19–26]. Mitral regurgitation has also been reported as a late complication of these procedures in association with recurrent dilatation [23–26]. Nishina et al. reported that improvements in LV size and function after plication in a rat ischemic cardiomyopathic model was transient because of residual scar remodeling and changes in LV geometry [23]. In a sheep model of MI, suturing a polyproplene mesh over the infarcted area prevented progressive LV expansion. This technique maintained normal ventricular geometry and heart function for 8 weeks despite substantial tissue losses [25]. Similarly, Leor et al. showed that implantation of a bioengineered fetal rat cardiomyocyte graft onto the surface of a MI attenuated ventricular dilatation [27]. The beneficial effects of this technique may be explained by the elastic properties of the bioengineered vascular smooth muscle graft. Like this bioengineered vascular smooth muscle graft, it is well known that PGCL scaffold has appropriate biodegradability and biocompatibility [26]. In the present study, we investigated the effects of tissueengineered cardiac patches consisting of BMMNC and PGCL scaffolds on attenuation of LV dysfunction and on induction of neovascularization in a rat model of infarcted heart. Rat BMMNCs were seeded onto PGCL scaffolds and implanted onto the epicardial surface over infarcted regions and adjacent normal heart. Four weeks after implantation, BMMNCs survived well in the scaffold, integrated into the host myocardium, and induced tissue preservation and neovascularization. Both patch groups had significantly improved LV function compared to control group. These favorable results can be explained as consequences of the cardioprotective effects induced by bioengineered scaffold and applied stem cells. PGCL scaffold alone, acting as a mechanical barrier, was able to prevent accelerated LV dilation after MI, and implanted BMMNC produced positive effects on both neovascularization and cardiomyocyte regeneration in ischemically jeopardized myocardium. With BMMNC, tissue ischemia was relieved and contractile dysfunction was attenuated by neovascularization. Matsubayashi et al. reported that repairing LV aneurysms with patches for a modified EVCPP composed of vascular smooth muscle cell-laden synthetic PCLA (sponge polymer of e-caprolactone-coL-lactide reinforced with knitted poly-L-lactide fabric) attenuated LV volume and systolic dysfunction in a rat MI model [28]. In this study, only the cell-seeded synthetic patches showed cardioprotective effects. This disparity with the previous result may be due to differences in the cell types applied, scaffold implantation time points after
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coronary artery ligation, and/or patch implantation methods. The type of cells chosen for implantation in treatment of MI is a vitally important question. Autologous cell implantation is intriguing in that it can avoid the problem of immunorejection. Obtaining autologous cells from bone marrow is attractive because marrow is easily obtained clinically and contains endothelial progenitor cells that could contribute to new blood vessel formation [28]. In addition, implantation of bone marrow-derived cells could induce regeneration of cardiac muscle and blood vessels in infarcted cardiac tissue, which is critical for recovery of heart function [31]. In clinical trials, intracoronary transplantation of bone marrow-derived cells into infarcted myocardium resulted in myocardial regeneration and neovascularization, and transendocardial transplantation of bone marrow-derived cells into infarcted myocardium safely promoted neovascularization and improved LV function [29–31]. In view of these past favorable results with BMMNC, we chose them for this study. A portion of the implanted BMMNCs seeded on PGCL scaffolds exhibited cardiomyocyte markers in this study. In previous reports, bone marrow stem cell transplanted into infarcted heart differentiated into smooth muscle cells, cardiomyocytes, and endothelial cells. Neovascularization was significantly greater in our BMMNC-seeded PGLC scaffold group than in the non-cell-seeded scaffold or sham-operated control groups. Neovascularization contributed to prolonged cell survival in the patches, and this may lead to improved collateral flow and augmented cardiomyocyte contractility and thus result in improvement of LV function. Even after the biodegradation of PGCL scaffold, this neovascularization in the ischemic zone may contribute to protection of the tissue against MI and to attenuation of subsequent progressive LV remodeling and LV systolic dysfunction. Implantation of organically structured stem cells on a biomaterial patch offers several advantages over direct injection of dissociated cells into infarcted cardiac tissue. In case of direct injection of dissociated cells, it is difficult to control shape and location in cardiac tissue [32], but with implantation of cells seeded on scaffolds, the polymeric scaffold provides a temporary support for the seeded cells until they produce their own extracellular matrix. This newly formed extracellular matrix around the cardiac patch maintains the structure and elasticity of the ventricular wall. Among biodegradable polymer scaffolds, PGCL scaffold is especially attractive for use in cardiac patches. A cardiac patch implanted onto the epicardial surface is under continuous contraction, so it needs to be sufficiently strong and elastic to resist damage from the contracting myocardium. The PGCL scaffolds used in this study were found to be highly elastic and flexible in mechanical tests, and they proved to be suitable in the mechanically dynamic environment of the heart. The combined advantages of PGCL scaffolds and BMMNC, with their vasculocardiomyogenic properties, were able to
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attenuate progressive LV dysfunction associated with MI in the rat model used in this study. 5. Conclusion BMMNC-seeded PGCL scaffold implantation effectively attenuated LV remodeling and LV systolic dysfunction in a rat myocardial infarction (MI) model. PGCL scaffold served as a mechanical barrier against progressive LV dilation in infarcted areas, and seeded BMMNCs survived, integrated into the myocardium, and effectively induced neovascularization. A portion of the implanted stem cells differentiated into cardiomyocytes. By this mechanism, BMMNC-seeded PGCL scaffold supplied contractile functioning cardiomyocytes, effectively reduced the ischemic area, and attenuated LV dilation, ultimately improving LV systolic function. The stem cell-seeded biocompatible scaffold can be applied as an alternative therapeutic modality for medically intractable advanced heart failure, especially for failure associated with MI. Acknowledgments This research was supported by a grant SC13121 and SC3150 [Dr. YB Park] from Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea. References [1] Zammaretti P, Jaconi M. Cardiac tissue engineering: regeneration of the wounded heart. Curr Opin Biotechnol 2004;15:430–4. [2] Klug MJ, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embryonic stem cells from stable intracardiac grafts. J Clin Invest 1996;98:216–24. [3] Koh GY, Soonpaa MH, Klug MG, Pride HP, Copper BJ, Zipes DP, et al. Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs. J Clin Invest 1995;96:2034–42. [4] Li RK, Jia ZQ, Weisel RD, Mickle DA, Zhang J, Mohabeer MK, et al. Cardiomyocyte transplantation improves heart function. Ann Thorac Surg 1996;62:654–60. [5] Li RK, Jia ZQ, Weisel RD, Merante F, Mickle DA. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol 1999;31:513–22. [6] Jain M, DerSimonian H, Brenner DA, Ngoy S, Teller P, Edge AS, et al. Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation 2001;103:1920–7. [7] Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7:430–6. [8] Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 2001;104:1046–52. [9] Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–5.
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