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Ann Thorac Surg 2004;78:303-307
© 2004 The Society of Thoracic Surgeons
a Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
b Department of Medicine, Duke University Medical Center, Durham, North Carolina, USA
c Department of Medicine and Physiology, University of Minnesota, Minneapolis, Minnesota, USA
Accepted for publication June 5, 2003.
* Address reprint requests to Dr Taylor, 7-112 BSBE, University of Minnesota, 312 Church St, SE, Minneapolis, MN 55455, USA
e-mail: dataylor{at}umn.edu
Abstract
PURPOSE: Currently, cells are transplanted into injured myocardium either through thoracotomy for open surgical delivery or through catheterization for endoventricular or intracoronary delivery; both methods have limitations. Open surgical delivery limits the potential patient population, whereas catheter-based delivery limits the ability to visualize the injection site and confirm delivery of the cells to the appropriate region. In this study, we examine the feasibility of cell transplantation into myocardium using a minimally invasive thoracoscopic approach.
DESCRIPTION: Seven swine underwent thoracoscopic cell transplantation. Using a prototype injection device, approximately 10 million myoblasts were injected into the anterior, lateral, posterior, and apical regions of myocardium. Animals were recovered up to 7 days, and after euthanasia, hearts were explanted for histology.
EVALUATION: All seven swine had successful delivery of myoblasts into the defined injection sites, as confirmed by analysis of an operative video, magnetic resonance imaging of iron-oxide–labeled cells, and histologic examination.
CONCLUSIONS: Thoracoscopic cellular cardiomyoplasty is feasible and allows the surgeon the benefits of direct visualization of the cell injection while minimizing morbidity associated with open cell delivery.
| Doctor Taylor discloses that she has a financial relationship with Bioheart, Inc.
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Cell transplantation to repopulate injured myocardium has shown to be an effective therapy for improving both systolic and diastolic ventricular function preclinically [1–3]. Consequently, both in the United States and Europe, safety and efficacy trials are under way to evaluate cell therapy as a new tool for cardiac repair [4, 5]. Current delivery methods are either through an open surgical approach during coronary artery bypass grafting or left ventricular assist device insertion; or through catheter-based delivery. Each of these methods has benefits and limitations. Percutaneous catheter delivery of cells has the benefit of being minimally invasive but lacks direct visualization of the injection region and precludes delivery to thin, scarred regions. Open surgical delivery necessitates a sternotomy or thoracotomy but allows precise delivery of cells to the desired region. For editorial comment, see page 14
By using a video-assisted thoracoscopic (VATS) ap-proach, we set forth to deliver cells to distinct areas of the heart with limited acute morbidity, while obtaining the benefits of precise delivery under direct visualization. Several limitations to video-assisted cell transplantation have been theorized: primarily a limited access to some areas of the myocardium—especially the posterior wall—and a difficulty in delivering cells to the beating heart (even under direct visualization). We conducted this experimental study to investigate the technical feasibility of VATS-based cellular cardiomyoplasty and to address those potential concerns.
Material and methods
All animals used received humane care in compliance with Duke University Institutional Animal Care and Use Committee guidelines and in compliance with "Guide forthe Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1985).
Technique
Seven swine (40 to 50 kg) were anesthetized with ketamine (10 mg/kg intramuscularly) and acepromazine (2 mg/kg intramuscularly) and intubated in the right main stem bronchus under fluoroscopic guidance. General anesthesia was maintained with isoflurane (2% to 5%). A 10-mm thoracoport (U.S. Surgical Corporation, Norwalk, CT) was inserted in the seventh intercostal space mid-axillary line. A 30-degree thoracoscope (Stryker Corporation, Stryker Endoscopy, San Jose, CA) was introduced through this port into the chest. Under direct visualization, a 5-mm port was inserted in the eighth intercostal space anterior axillary line. After placing the port, the trochar was removed to allow the introduction of both endoscopic shears and grasper, as shown in Figure 1, A.
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Two animals were injected with myoblasts that were additionally labeled with Feridex superparamagnetic ironoxide particles (SPIO; Berlex Laboratories, Montville, NJ), which allows detection of iron labeled cells in vivo by magnetic resonance imaging (MRI) [6]. These animals survived for 7 days before MRI.
Cell culture technique
Tissue from a 500 to 700 mg hindlimb biopsy of a heterologous swine was mechanically dissected into 1 mm3 pieces and washed once in sterile phosphate buffered saline (PBS), before resuspension and plating in myoblast growth medium, consisting of 10% fetal bovine serum and 0.5% gentamicin (10 mg/mL). The tissue fragments were triturated after 3 days, and the tissue was removed after 5 days. The myoblasts were expanded in myoblast growth medium at densities of less than 70% for 14 days (2 to 4 passages). Cells were incubated for 4 hours with 10 µg/mL of the fluorescent marker 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO), before harvest for injection. For injection, 1 x 107 myoblasts per animal were trypsinized, washed twice in PBS, resuspended in 1.0 mL saline for injection, and loaded into the injection device.
For Feridex labeling, cells were incubated with Feridex at 10 µg/mL and Lipofectamine 2000 (Gibco-Invitrogen Corp, Carlsbad, CA) at 3 µg/mL in OptiMEM medium (Gibco) for 24 hours, followed by extensive washing in PBS.
Cell viability
After passage through the injection device, cell viability was assessed after passing through the injection device by a standard Trypan blue exclusion. The number of viable cells (Trypan blue exclusion) was counted under a microscope using a hemacytometer and expressed as a percentage of the total number of cells (Trypan blue uptake and Trypan blue exclusion).
Magnetic resonance imaging
Two animals were studied with MRI 7 days after injection of SPIO-labeled myoblasts into the posterior wall. Magnetic resonance imaging was performed using a 1.5 Tesla clinical scanner (Siemens AG, Munich, Germany). Electrocardiography gated cine MR images were acquired of the whole left ventricle using a steady-state free precession (SSFP) pulse sequence, obtaining both long and short axis views. Imaging measurements were as follows: Field of view 280 x 254 mm, echo time 1.86 ms, repetition time 33.39 ms, in plane resolution 1.09 x 1.09 mm, slice thickness 5 mm.
Histology
After explanting the hearts, the cell-injected areas of the myocardium were identified. These areas were embedded in optimum cutting temperature freezing medium, frozen in isopentane cooled liquid nitrogen, and cut on a cryostat in 5-micron thick serial sections. Sections were analyzed for the presence of DAPI and iron-oxide–labeled cells. Iron was detected with a standard Prussian blue iron stain (Sigma) according to the manufacturer's instructions.
Results
Passage of cells through the injection device did not influence viability, which was measured at more than 98% for three different cell populations. Marker dye was identified in the anterior, lateral, posterior, and apical left ventricular wall after explant (Fig 2). Successful delivery of cells to all 7 animals was confirmed by histologic identification of fluorescently labeled nonnative cells in the injection locations of each heart (Fig 3). Cine MRI of 2 animals showed the presence of iron-oxide–labeled myoblasts in the posterior LV wall 7 days after injection (Fig 4). In addition, all seven swine successfully tolerated the procedure and survived until the euthanasia point either 4 hours or 7 days postoperatively.
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No hemodynamically significant arrhythmias were seen when manipulating the heart; however, some premature ventricular contractions could be documented, particularly after the heart stabilization procedure. Of note, when incising the pericardium, it was critical to create a sufficiently large window not only to be able to maneuver the myocardium and deliver cells to the appropriate areas but also to prevent atrial herniation, which temporarily occurred in one animal until the window was enlarged.
Comment
As research in cell therapy has progressed, the actual injection process has come under more scrutiny due to the realization of the importance of precise delivery of cells and the difficulty in generating vigorous engraftment. Furthermore, it has become clear the positive functional outcome is directly related to the number of cells delivered [7]. To provide a minimally invasive surgical technique that allows maximum cell delivery, we developed a VATS approach. In this feasibility study we show that by utilizing a thoracoscopic approach, cells can be effectively transplanted into host myocardium at all major potential infarct zones of the left ventricle, including the posterior wall, while still maintaining a precise direct visual delivery.
Of the currently described available delivery modalities, open chest cell delivery, usually as an adjunct to coronary artery bypass graft surgery, has received the most study. This open technique has had early success in patients who receive concurrent revascularization [4]. However, this need for revascularization has also confounded interpretation of any functional improvement seen in these patients. Thoracoscopic approaches provide the ability to deliver cells to injured myocardium without the need for sternotomy. The ability to deliver cells as an independent procedure, without coronary artery bypass graft surgery, should allow investigators to pinpoint functional improvement in patients after only cell therapy.
The other major technique currently used for delivery of cells is a catheter-based approach, in which an injection catheter is introduced either into the left ventricle, or through the femoral vein into the coronary sinus and great cardiac vein for access to the left ventricle. These techniques require either fluoroscopic guidance or ventricular mapping technology to define infarct zones, but still are unable to provide any direct visualization [8]. Further, under fluoroscopic guidance, precise localization of infarct zones is difficult and engraftment potentially suffers, as opposed to direct surgical injection, which has the potential to yield vigorous deposits in precise areas (Fig 3, A). Similarly, ventricular electrophysiologic mapping requires interrogation of dozens of locations on the endocardium and takes a significant amount of time. Finally, catheter-based delivery is limited to areas greater than 5 mm in thickness because of possible perforation, which limits the patient population to those who have not undergone significant wall thinning.
Thoracoscopic cell transplantation could offer the cardiac surgeon an excellent opportunity to provide a select group of patients, for whom revascularization is not indicated, the option to receive the benefit of cell therapy. Video-assisted thoracoscopic cell delivery offers clear benefits over both open surgical and catheter-based delivery. Of note, our study clearly only examines feasibility, and lacks any controlled comparison with other injection techniques regarding cell engraftment or effect on functional indicators. Our intent was to investigate the ability to successfully deliver cells by this technique to all areas of the left ventricle at risk for infarction. Ongoing preclinical studies are aimed at comparing various delivery techniques (open, catheter-based, and VATS) and their effect on cardiac physiology, cell engraftment, and morbidity.
Disclosures and freedom of investigation
The authors had full control of the design of the study, methods used, outcome measures, analysis of data, and production of the written report.
Acknowledgment
This work was supported in part by NHLBI/National Institutes of Health awards to Dr Taylor (R-01 HL-57988, HL-63346, HL-63703).
Footnotes
The Society of Thoracic Surgeons, the Southern Thoracic Surgical Association, and The Annals of Thoracic Surgery neither endorse nor discourage use of the new technology described in this article.
References
This article has been cited by other articles:
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  H. C. Ott, J. Brechtken, C. Swingen, T. M. Feldberg, T. S. Matthiesen, S. A. Barnes, W. Nelson, and D. A. Taylor Robotic minimally invasive cell transplantation for heart failure J. Thorac. Cardiovasc. Surg., July 1, 2006; 132(1): 170 - 173. [Full Text] [PDF]
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 J. C Chachques, C. Salanson-Lajos, P. Lajos, A. Shafy, A. Alshamry, and A. Carpentier Cellular Cardiomyoplasty for Myocardial Regeneration Asian Cardiovasc Thorac Ann, September 1, 2005; 13(3): 287 - 296. [Abstract] [Full Text] [PDF]
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