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Ann Thorac Surg 2002;74:1568-1575
© 2002 The Society of Thoracic Surgeons

Original article: cardiovascular

Significant improvement of heart function by cotransplantation of human mesenchymal stem cells and fetal cardiomyocytes in postinfarcted pigs

Jiang-Yong Min, MDa, Matthew F. Sullivan, BSa, Yinke Yang, MD, PhDa, Jian-Ping Zhang, MDa, Kimber L. Converso, BSa, James P. Morgan, MD, PhDa, Yong-Fu Xiao, MD, PhDa*

a Stem Cell Research Laboratory, The Charles A. Dana Research Institute and Harvard-Thorndike Laboratory, Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA

Accepted for publication June 26, 2002.

* Address reprint requests to Dr Xiao, Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Boston, MA02215, USA.
e-mail: yxiao{at}caregroup.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
BACKGROUND: Viable cardiomyocytes after myocardial infarction (MI) are unable to repair the necrotic myocardium due to their limited capability of regeneration. The present study investigated whether intramyocardial transplantation of human mesenchymal stem cells (hMSCs) or cotransplantation of hMSCs plus human fetal cardiomyocytes (hFCs; 1:1) reconstituted impaired myocardium and improved cardiac function in MI pigs.

METHODS AND RESULTS: Cultured hMSCs were transfected with green fluorescent protein (GFP). Six weeks after MI induction and cell transplantation, cardiac function was significantly improved in MI pigs transplanted with hMSCs alone. However, the improvement was even markedly greater in MI pigs cotransplanted with hMSCs plus hFCs. Histological examination demonstrated that transplantation of hMSCs alone or hMSCs plus hFCs formed GFP-positive engrafts in infarcted myocardium. In addition, immunostaining for cardiac {alpha}-myosin heavy chain and troponin I showed positive stains in infarcted regions transplanted with hMSCs alone or hMSCs plus hFCs.

CONCLUSIONS: Our data demonstrate that transplantation of hMSCs alone improved cardiac function in MI pigs with a markedly greater improvement from cotransplantation of hMSCs plus hFCs. This improvement might result from myocardial regeneration and angiogenesis in injured hearts by engrafted cells.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
As a result of limited myocyte regenerative capabilities, injured myocardium cannot prevent the onset and evolution of ventricular dysfunction. Although limited regeneration of cardiomyocytes has recently been reported in human infarcted hearts [1], it is generally acknowledged that dead myocardium is replaced by nonfunctional fibrous tissue. High morbidity of ischemic cardiac dysfunction and shortage of donor hearts demand a constant search for new approaches to treat heart failure. Cell transplantation, including use of AT1 [2], fetal [3] or neonatal cardiomyocytes [4], adult porcine [5] or rat myocytes [6], satellite cells [7], and bone marrow cells [8–10], has been demonstrated to be of therapeutic value for the repair of damaged myocardium in animal models. More recently, we successfully transplanted mouse embryonic stem (ES) cells into postinfarcted rat hearts [11, 12], which resulted in functional improvement for 32 weeks after the induction of myocardial infarction (MI). ES cells may be an important source for cell therapy due to their great plasticity and capability of differentiation into all cell types of the body. However, political and ethical debates on human embryonic stem cells limit the progress of their therapeutic applications. Therefore, the feasibility of using human adult stem cells, such as human mesenchymal stem cells (hMSCs), for cell therapy is of great clinical significance.

Bone marrow contains a population of rare progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells, including cardiac muscles [13]. A recent study showed that the implanted bone marrow cells differentiated into myocytes and coronary vessels, ameliorating the function of the injured heart [9]. However, in order to provide important clinical insights for cell therapy, key experiments are required to be carried out in large animals. A recent study demonstrated that transplantation of hMSCs alone improved regional function in infarcted swine myocardium [14]. Therefore, the present study was undertaken to investigate whether transplantation of hMSCs alone could improve global heart function and whether cotransplantation (1:1) of hMSCs plus human fetal cardiomyocytes (hFCs) could produce a greater improvement of cardiac function in MI pigs. Hemodynamic evaluation of left ventricular function and histologic evidence of engrafted cells in injured myocardium were evaluated and compared in MI pigs with or without cell transplantation.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
Cell culture and preparation
The hMSCs and hFCs (19 weeks) were obtained from BioWhittaker Inc. (Walkersville, MD) and maintained with the method previously described [15]. Briefly, a Bullet-kit containing mesenchymal growth supplements was used to culture cells. Before cell transplantation, hMSCs were transfected with plasmids of the hCMVIE promoter/enhancer driving green fluorescent protein gene (5.7 kb) by the method described previously [11] to identify the survival of engrafted cells. Approximately 3 x 105 of hMSCs were plated in 100-mm dishes and cultured to obtain approximately 90% confluence on the day of transfection. Two days after GFP transfection, cultured hMSCs were trypsinized and centrifuged. Collected hMSCs and hFCs were resuspended in the cultured medium for cell transplantation with a concentration of 107 cells/mL. The proportion of GFP-positive hMSCs was about 70 ± 5% (n = 4), which were counted under fluorescent microscopy.

Experimental animals and surgical preparations
MI was performed in male Yorkshire pigs with a body weight of approximately 15 kg. The animals were sedated with ketamine (15 mg/kg, IM) and thiopentathal (5 mg/kg, IV). After tracheal intubation, animals were ventilated with approximately 2% isoflurane at a rate of 12 breaths/minute (Hallowell EMC Model 2000; Veterinary Anesthesia Ventilator, Pittsfield, MA). Electrocardiogram and respiration were monitored by a multiple-channel recorder (Portal Systems Inc., Beaverton, OR). The right femoral artery was isolated and cannulated with an introduction sheath. Through the catheter sheath, a 7-Fr pig-tailed catheter was retrogradely advanced into the left ventricle. The catheter was connected to a pressure transducer, and intraventricular pressure was recorded by a chart-strip recorder. The LVSP, LVEDP, +dP/dt, and -dP/dt were measured to evaluate ventricular function.

The heart was exposed by means of a left thoracotomy through the fourth and fifth intercostal space. The distal end, just below the third diagonal, of the left anterior descending coronary artery was ligated. Five minutes after ligation, 7 x 106 cells of either hMSCs plus hFCs (1:1, 107 cells/mL) or hMSCs alone (107 cells/mL) were injected into the border area of ischemic myocardium. Control MI hearts received the same MI operation, but were injected with the same volume of the cell-free medium. Antibiotics (Cefazolin, 35 mg/kg, IM) and analgesics (Buprenex, 0.03 mg/kg, IM) were administered shortly after surgery. Cefazolin (35 mg/kg) was maintained daily for 5 days after operation. Cyclosporine A, 15 mg/kg, was given orally every other day until the animals were sacrificed. This dosage should be able to reduce immunoreaction to engrafted organs in heart-lung transplant patients and in pigs with allogeneic heart transplantation [16, 17].

Assessment of cardiac function and myocardial blood flow
Ventricular function was assessed by echocardiography as described previously [12]. Briefly, a commercially available echocardiographic system equipped with a 12.5-MHz probe (Agilgent Sonos 5500 [Agilent Technologies, Palo Alto, CA]) was used and the results were analyzed from data recorded on an optical disk [11]. Direct measurement of hemodynamics was conducted by intraventricular catheterization in MI pigs before ligation (baseline values), and 1 hour and 6 weeks after MI induction and cell implantation.

Six weeks after cell transplantation, stable isotope-labeled microspheres (BioPAL Inc., Worcester, MA) were used to determine coronary blood flow [18] in anesthetized MI pigs under resting condition or with pacing stress by electric stimulation, 180 beats/minute. In brief, a set of microspheres (2 x 106) were diluted in 3 mL of sanSaLine saline (BioPAL Inc.) and injected into the left atrium over 30 seconds. Reference blood samples were withdrawn by using a syringe pump at a constant rate of 5 mL/min through the femoral artery to calculate absolute myocardial blood flow. Finally, the heart was excised and regional myocardial blood flow was determined by BioPAL Inc. [18, 19], where collected heart tissues and blood samples were exposed to a field of neutrons.

Morphology and histology of infarcted myocardium
Subsets of animals were sacrificed after assessment of hemodynamics and blood flow 6 weeks after MI. The hearts were quickly removed, and selected tissues from the free wall of the left ventricle, including infarct and periinfarct regions, were embedded in tissue freezing medium (Fisher Scientific, Fair Lawn, NJ). Frozen sections (8 µm in thickness) of left ventricular tissue were made for identification of implanted cells and for immunofluorescent staining. Other hearts were fixed in 10% formalin overnight. The cardiac tissues were paraffin embedded and sectioned at 5-µm thickness for hematoxylin and eosin staining. GFP-positive spots under fluorescent microscopy represented the presence of engrafted cells in injured myocardium. Immunostaining for {alpha}-myosin heavy chain ({alpha}-MHC) and cardiac troponin I (cTnI) was used to identify the survival and differentiation of engrafted hMSCs and hFCs. Frozen sections were washed three times in phosphate-buffered saline (PBS) and incubated with Cy3-conjugated goat anti–mouse IgG antibody (Sigma, St. Louis, MO) for 45 minutes. Nonspecific binding was blocked by incubation with 1% bovine serum in remaining sections. Different frozen sections were stained immunohistochemically with a mouse monoclonal anti-GFP antibody (Zymed, San Francisco, CA), a goat polyclonal IgG anti-cTnI antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), or a mouse anti-{alpha}-MHC monoclonal antibody (Berkeley Antibody Co, Richmond, CA) for 60 minutes. After washing with PBS, sections were incubated with a rabbit anti–goat conjugated rhodamine IgG (for cTnI) or a goat anti–mouse conjugated fluorescein IgG (for {alpha}-MHC and GFP) antibody (Pierce Chemical Co, Rockford, IL). Finally, fluorescent staining for {alpha}-MHC and cTnI were detected and photographed under fluorescent microscopy.

Data analysis
All values are presented as means ± SE. The data collected before and after cell transplantation were compared by the paired Student’s t test in each group. Analysis of variance was used for the comparison of the differences among the data delivered from more than two groups. A p < 0.05 was considered as significantly different.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
Twenty-five animals received permanent ligation of the left coronary artery just below the third diagonal branch. Five animals died of lethal ventricular arrhythmias within 24 hours after MI operation. The study was comprised of the following groups: MI pigs transplanted with hMSCs alone (MI-hMSCs, n = 6); MI pigs cotransplanted with hMSCs and hFCs (MI-hMSCs+hFCs, n = 7); and MI control pigs injected with an equivalent volume of the cell-free culture medium (MI-control, n = 7). Baseline ventricular function before induction of MI assessed by hemodynamic measurements was not significantly different among the three groups. Myocardial infarction decreased cardiac function reflected by reduction of the left ventricular systolic pressure (LVSP), peak rate of the left ventricular systolic pressure rise (+dP/dt), and peak rate of the left ventricular systolic pressure fall (-dP/dt). Additionally, the left ventricular end-diastolic pressure (LVEDP) was elevated in all infarcted animals compared with their pre-MI values. Six weeks after MI operation, cell transplantation significantly improved the ventricular function by reducing the LVEDP and increasing LVSP, +dP/dt, and -dP/dt (Fig 1; Table 1). The beneficial effects of cell transplantation on cardiac function were even greater in MI pigs cotransplanted with hMSCs plus hFCs. The increased improvement of cardiac function in cotransplanted animals was persistent when the MI hearts were paced at a rate of 180 beats/minute (Table 1).



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  		Fig 1. Hemodynamic measurements in postinfarcted porcine hearts before ligation (Baseline), and 1 hour and 6 weeks after myocardial infarction (MI). Cell transplantation of hMSCs alone or hMSCs plus hFCs improved the ventricular function compared with the MI control animals. There is a trend of greater beneficial effects on ventricular function with cotransplantation of hMSCs plus hFCs compared with transplantation of hMSCs alone. MI-Control = postinfarcted pigs with transplantation of the cell-free medium (n = 7); MI-hMSCs = postinfarcted pigs with transplantation of hMSCs alone (n = 6); MI-hMSCs+hFCs = postinfarcted pigs with cotransplantation of hMSCs plus hFCs (n = 7). (a) LVSP = the left ventricular systolic pressure; (b) LVEDP = the left end-diastolic pressure; (c) +dP/dt = the peak rate of pressure rise; (d) -dP/dt = the peak rate of pressure fall. *p < 0.05, **p < 0.01 versus MI-Control at 6 weeks after MI; #p < 0.05 versus MI-hMSCs 6 weeks after MI. (hFCs = human fetal cardiomyocytes; hMSCs = human mesenchymal stem cells.)

 

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  		Table 1. Hemodynamic Measurements in Postinfarcted Pig Hearts at 6 Weeks After Cell Transplantation

 
The baseline echocardiographic parameters were similar among the animals with or without cell transplantation (data not shown). Six weeks after MI operation, left ventricular contractility was decreased in MI pigs injected with the cell-free medium (Table 2). Cell transplantation significantly improved ventricular function, reflected by an increase of endocardial fractional shortening, mid-wall fractional shortening, stroke volume, and cardiac index. The beneficial effects were more profound in MI pigs that received cotransplantation of hMSCs plus hFCs than in animals transplanted with hMSCs alone.


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  		Table 2. Echocardiographic Measurements of Ventricular Function In Vivo 6 Weeks After Cell Transplantation in Porcine Hearts

 
Histologic staining of myocardial sections with hematoxylin and eosin showed fibrosis without regenerated cell islets in infarcted myocardium injected with the cell-free medium 6 weeks after MI (Fig. 2b, 2c). Engrafted cells were diversely distributed in infarcted areas transplanted with hMSCs alone (Fig 2e, 2f) and hMSCs plus hFCs (Fig 2h, 2i). In addition, we did not find pronounced evidence of immunorejection of engrafted cells (eg, significant infiltration of either lymphocytes or macrophages in the engrafted area with hMSCs alone or hMSCs plus hFCs). A possible explanation is that the pigs were treated with the immuno-inhibitor cyclosporine, and the small amount of transplanted cells might not be able to trigger immunorejection. Positive GFP spots observed under fluorescent microscopy demonstrated the survival of engrafted cells in myocardium transplanted with hMSCs alone (Fig 2d) or hMSCs plus hFCs (Fig 2g). Furthermore, the intensity of immunostaining for {alpha}-cardiac myosin heavy chain ({alpha}-MHC; Fig 3 left column) and cardiac troponin I (cTnI; Fig 3 right column) was much higher in infarcted myocardium with transplantation of hMSCs alone or hMSCs plus hFCs than with injection of the cell-free medium. Double staining for GFP and cTnI of injured myocardium cotransplanted with hMSCs plus hFCs was shown in Fig 4, which further confirms that implanted cells could differentiate into cardiac-like cells.



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  		Fig 2. Morphology of hematoxylin & eosin (H&E) staining of normal porcine myocardium (a), and infarcted myocardium with medium injection (b and c). Green fluorescent protein–positive clusters sectioned from myocardial infarction pig hearts with transplantation of hMSCs alone and cotransplantation of hMSCs plus hFCs are shown in d and g, respectively. The H&E staining of infarcted porcine myocardium showed cell grafts within the infarcted zone with transplantation of hMSCs alone (e and f) and cotransplantation of hMSCs plus hFCs (h and i). The arrows in b, e, and h point to the areas corresponding with the magnification in c, f, and i. (hFCs = human fetal cardiomyocytes; hMSCs = human mesenchymal stem cells.)

 


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  		Fig 3. Positive immunofluorescent staining to {alpha}-MHC and cTnI were found in normal (a and b, respectively) and postinfarcted myocardium within the infarcted zone transplanted with hMSCs alone (e and f) and cotransplantation with hMSCs plus hFCs (g and h, respectively), but not in injured porcine myocardium with medium injection (c and d, respectively). The results were obtained from different animals for fluorescent labeling of {alpha}-MHC and cTnI (x200). ({alpha}-MHC = {alpha}-myosin heavy chain; cTnI = cardiac troponin I; hFCs = human fetal cardiomyocytes; hMSCs = human mesenchymal stem cells.)

 


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  		Fig 4. Double staining for GFP and cTnI of injured myocardium cotransplanted with hMSCs plus hFCs. a and b show the staining of GFP by a monoclonal anti-GFP antibody and of cTnI by a polyclonal anti-cTnI antibody, respectively. The merger (c) of GFP and cTnI staining demonstrates that engrafted GFP-positive cells differentiated into cardiac myocytes (x200). (CTnI = cardiac troponin I; GFP = green fluorescent protein; hFCs = human fetal cardiomyocytes; hMSCs = human mesenchymal stem cells.)

 
In addition to evaluation of cardiogenesis in infarcted myocardium with cell transplantation, regional blood flow of the myocardium was measured by neutron microspheres. Six weeks after MI induction, resting blood flow was significantly decreased in control MI myocardium injected with the cell-free medium (Fig 5). Compared with control MI hearts, cell transplantation increased resting blood flow in infarcted myocardium. The increase was even greater in pigs cotransplanted with hMSCs plus hFCs than in animals transplanted with hMSCs alone. Furthermore, the beneficial effects on blood flow were also observed under the pacing-induced stress condition in MI hearts with cell transplantation. Again, cotransplantation of hMSCs plus hFCs produced a greater increase in blood flow during pacing than with hMSCs alone.



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  		Fig 5. Blood flow measurements with the neutron microsphere technique in postinfarcted porcine hearts at resting condition (a) and with pacing stress (b). MI-Control = postinfarcted pigs with transplantation of the cell-free medium (n = 7); MI-hMSCs = postinfarcted pigs with transplantation of MI-hMSCs (n = 6); MI-hMSCs+hFCs = postinfarcted pigs with cotransplantation of hMSCs plus hFCs (n = 7). *p < 0.05; **p < 0.01 versus MI-Control; #p < 0.05 versus MI-hMSCs. (hFCs = human fetal cardiomyocytes; hMSCs = human mesenchymal stem cells.)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
It has been recognized that cardiomyocyte transplantation into scarred tissues after MI improves myocardial function [3–6, 20]. Grafted cardiomyocytes foster electrical pathways with host counterparts through the gap junction [21, 22]. Recently, Orlic and associates [9] reported that transplanted lineage-negative bone marrow cells regenerated 68% of myocardium in the infarcted portion of the left ventricle within 9 days after cell transplantation, and significantly improved cardiac hemodynamics. Our previous study demonstrated that transplanted embryonic stem cells survived in injured rat myocardium and improved global cardiac function up to 32 weeks [11, 12]. However, application of human embryonic stem cells for cell therapy encounters strong political and ethical challenges. In addition, transplantation of animal embryonic stem cells into diseased human hearts with application of immunosuppressive agents has not been tested yet and is probably unacceptable for heart failure patients. Therefore, human adult stem cells are alternative cell sources for clinical application. In this study, we demonstrated that intramyocardial transplantation of hMSCs improved infarcted heart function, and the improvement was even greater in MI pigs cotransfected with hMSCs and hFCs. Mild attenuation of LV remodeling after cell transplantation, which is reflected by an increase in posterior relative wall thickness and anterior relative wall thickness with echocardiography, might partially contribute to enhancement of myocardial contractility. However, it cannot explain the greater improvement of cardiac function in the MI pigs with cotransplantation of hMSCs plus hFCs than with transplantation of hMSCs alone, because there was no significant increase in wall thickness in the cotransplanted group.

The relative accessibility of bone marrow and the possibility for autologous transplantation make bone marrow cells (BMCs) a potential candidate for cell therapy after myocardial infarction. Mesenchymal stem cells (MSCs) can be isolated from bone marrow and are able to participate in the renewal of many tissues, because MSCs retain the potential to differentiate into adipocytes, myocytes, muscles, chondrocytes, and fibroblasts [13, 23, 24]. In the present study, transplantation of hMSC alone improved cardiac function in infarcted pigs 6 weeks after MI induction and cell implantation, but cotransplantation of hMSCs plus hFCs produced greater functional recovery. Engrafted hMSCs or hMSCs plus hFCs not only survived in injured porcine myocardium, but also generated new myocardium evidenced by positive staining for {alpha}-MHC and cTnI. It has been demonstrated that BMC transplantation regenerated infarcted myocardium in mice [9] and formed cardiac-like muscle cells in cryoinjury-derived ventricular scar tissue in rats [8]. However, only 5-azacytidine- (which has been demonstrated to induce BMC differentiation into cardiomyocytes in vitro [25]) treated BMC-transplanted hearts had higher systolic and developed pressures than those of the control hearts [8]. No difference was found among control and non-5-azacytidine-treated BMC transplanted hearts. Our present data suggest that the improvement of ventricular function, at least partially, results from new cardiomyocytes differentiated from engrafted hMSCs cells. One explanation for the lesser improvement of cardiac function in MI hearts transplanted with hMSCs alone is that engrafted hMSCs may not yield sufficient myocyte numbers to repair the injured myocardium. In contrast, implanted hFCs might survive in injured myocardium and form more new myocardium to further improve heart function in MI pigs with cotransplantation of hMSCs plus hFCs. These results are consistent with the findings by Li and colleagues that fetal cardiac cells had superior growth potential to improve heart function compared with adult or pediatric cardiomyocytes [26], and cardiomyocyte transplantation [27]. Further quantitative measurements of regenerating myocardium are surely required.

Therapeutic myocardial angiogenesis has been widely accepted for treating myocardial ischemia by the use of proangiogenic growth factors to induce the development of new blood vessels in the myocardium at risk [28, 29]. An increase of angiogenesis resulted in a significant improvement of regional blood flow and global cardiac function in chronically ischemic porcine myocardium [30]. In the present study, the blood supply to the ischemic territory, as assessed by neutron microsphere method, was improved by the transplantation of hMSCs alone and hMSCs plus hFCs. Engrafted cells may provide the cell source for formation of new blood vessels and release vascular endothelial growth factor to induce new capillary formation and growth in injured myocardium. It has been reported that transplantation of BMCs with or without 5-azacytidine treatment induced angiogenesis in injured rat hearts [8]. In addition, transplantation of early-differentiated cells from mouse embryonic stem cells significantly increased the densities of blood vessels and improved cardiac function in post-MI mice. Adding a vascular endothelial growth factor gene to the cells further enhanced the beneficial effects of cell transplantation on postinfarcted hearts [31]. Therefore, neovascularization caused by engrafted cells may affect the long-term survival of cardiac engrafts in injured myocardium. The more pronounced increase in blood flow within the ischemic myocardium cotransplanted with hMSCs plus hFCs could partially explain the greater improvement of myocardial function in vivo assessed by hemodynamic and echocardiographic measurements. The underlying mechanism for higher blood flow in injured myocardium cotransplanted with hMSCs plus hFCs is not clear. However, it is possible that the larger amount of surviving engrafted cells plus differentiated cardiac-like cells from hMSCs and hFCs may deliver strong signals for the requirement of more blood flow to the cell-implanted myocardium to provide enough oxygen and nutrition.

It has been shown that mesenchymal stem cells (MSCs) are well tolerated when transplanted to humans and animals [32]. After transplantation, the differentiation appears to be controlled by local factors in the respective tissues. When cotransplanted together with hematopoietic cells in a stem cell transplantation setting, MSCs appear to enhance engraftment of the hematopoietic cells as well as reduce the incidence and severity of graft-versus-host disease. As we lack the results of transplantation of hFCs alone, we do not know whether cotransplantation of hMSCs and hFCs is better than transplantation of hFCs alone. Therefore, additional experiments are required to determine the effects of transplantation of hFCs alone on cardiac function in infarcted pigs. However, our data do support that cotransplantation of hMSCs and hFCs produced a better improvement of cardiac function than transplantation of hMSCs alone in MI pigs.

We conclude that transplantation of hMSCs alone or hMSCs plus hFCs can regenerate injured myocardium and improve cardiac function in our MI porcine model. The functional improvement is greater in MI hearts cotransplanted with hMSCs plus hFCs. The better heart function in cotransplanted MI animals may result from more regenerated cardiomyocytes and a stronger angiogenesis effect caused by engrafted hFCs.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

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