Catheter system for cell implantation. This novel 8 Fr compatible system has three components: 1) injection guide catheter, 2) stiletto, and 3) injection needle catheter (Figure 1). The injection guide catheter delivers the needle device into the target lesion. The injection guide catheter has three lumens: two of these are for guidewires and both are of the monorail-type with two resin layers. The last lumen is for the injection needle catheter. The stiletto protects the lumen from damage and helps deliver the injection needle catheter to the lesion. The stiletto is made of stainless steel. Its distal end is radiopaque and covered with platinum coils. The rest of the stiletto is coated with polytetrafluoroethylene. The injection needle catheter consists of layers of polymer and stainless steel braiding, and is used for cell implantation. A 28 gauge curved nickel-titanium alloy needle is connected to the distal end of the shaft. The inner diameter is 0.40 mm and the outer is 0.56 mm. The double-guidewire technique is used to perform the implantation in this system. The procedure should be performed in a stepwise manner. One guidewire is inserted into the main vessel (Figure 2A) and the other is advanced into the side branch (Figure 2B). Next, the guide catheter is inserted along the first wire and advanced to the main vessel (Figure 2C), and the second guidewire lumen in the catheter is positioned such that the second guidewire passes through it (Figure 2D). The guidewires form a flat surface from which the needle projects vertically (Figure 2E), thus directing the injection toward the myocardium (Figure 2F). Based upon the length of the needle, the depth of injection is assumed to be 2-5 mm from the surface of the myocardium. Cells (0.2-0.4 cc) are then infused through the injection needle catheter.
Isolation and expansion of skeletal myoblasts. Primary skeletal myoblasts were isolated from Yorkshire swine (n = 8; 50-60 kg). Hind-limb muscles (1.5-2.0 g) were dissected free from connective tissues, and minced into pieces of approximately 1 mm3. Muscle samples were enzymatically dissociated according to the cell dispersion technique described by Blau and Webster.11 Before the intramyocardial injection, cells were labeled using a fluorescent cell-linker kit (PKH26-GL, Sigma-Aldrich Co., Vienna, Austria) according to the instructions of the manufacturer, as previously described.12
Transcoronary artery myocardial access. A transfemoral artery approach with an 8 Fr arterial sheath was used for the coronary angiographic procedure in swine under systematic anesthesia. The left main coronary artery was selectively engaged with an 8 Fr Hockey Stick PCI guiding catheter (Medtronic Zuma, Santa Rosa, California) and control angiography was performed. Two wires were inserted in the left anterior descending artery (LAD), one was placed in the diagonal branch and the other in the distal LAD. Next, the double-wire catheter was introduced into the LAD and the targeted areas in the myocardium were punctured. The injection needle catheter was advanced through the guide catheter, the myocardial tissue was punctured with the needle and the myoblasts were injected into the tissue.
Histology and immunohistochemistry. Animals (n = 8) were euthanized with an overdose of intravenous potassium chloride 40 mEq on Days 0 (n = 4) and 14 (n = 4). Accuracy of placement of the injected cells was assessed by correlating dye stains identified by gross examination using anatomic and fluoroscopic landmarks. Histological sections of the injection sites were divided into basal and apical subsegments of the anterior, lateral, anteroseptal and inferoseptal walls. Portions of porcine myocardium acquired from the injection site were frozen in Tissue Tek optimal cutting temperature compound and stored at -80 C for subsequent histological analysis. The ventricles were then cross-sectioned into three sections. From each section, 8 µm thick slides were prepared using a cryostat, and standard histological studies were performed with hematoxylin and eosin staining. The transplanted myoblasts were identified by fluorescence.
Animal care. All animal care procedures were conducted in accordance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).
The guide catheter was successfully delivered into the LAD in all animals. Cell injection using the injection needle catheter was performed after successful introduction of the guide catheter into the LAD. No complication was observed during the procedure. The time taken to complete the procedure was 56 minutes on average. No arrhythmia occurred during the cell injection. There were no events of death, cardiac tamponade or sustained arrhythmia during the period until the animals were euthanized.
The dye stains correlated 100% with the gross examination landmarks. Figure 3 shows the anatomical picture after the injection of the dye, revealing the extent of distribution of myoblast cells after a single injection (Figure 3A). Myoblast hyperplasia was observed 14 days after injection (Figure 3B). PKH26-positive myoblast cells were present at Days 0 and 14, as demonstrated by fluorescence microscopy (Figure 3C).
Previous studies focusing on the treatment of infarcted myocardium by cellular transplantation have demonstrated promising outcomes for this novel and effective therapeutic procedure.13-17 Although cellular transplantation is expected to improve the outcome of patients with myocardial infarction, the most appropriate cell delivery system has not yet been established. The candidate cell-delivery method must safely deliver an adequate number of cells to the damaged site. The intravenous mode was considered to be a potential candidate, however, it demonstrated low efficiency in delivery to the damaged site.18 The endocardial mode demonstrated safety and efficacy of cell delivery to damaged sites,19-21 but the system used at present requires equipment normally found only in catheterization laboratories, thus failing to meet two requirements of the ideal delivery system: user-friendliness and cost-effectiveness.
Our approach of intracoronary delivery using the conventional PCI technique offers a simple and novel cell-delivery system. Direct injection without surgical technique may maximize the deposition of infused cells within the region of interest. To the best of our knowledge, this is the first approach of its kind for injection of cells into damaged myocardium using the conventional PCI technique. We hypothesize that this novel cellular cardiomyoplasty system reduces the target tissue, thereby improving cell dispersion and engraftment. This catheter system consists of a double-wire catheter and an injection needle catheter. The double-wire catheter safely guides the needle device so that it is positioned precisely at the damaged myocardium. Next, two wires are advanced into branches of the peripheral vessels, allowing the needle tip to be reliably directed toward the myocardium. The injection needle catheter has a unique stiletto structure to protect the needle tip and a design that allows cell activity to be maintained. The main characteristic of this system is its easy and simple handling using the PCI technique. No additional system or equipment is needed to handle this catheter. In the present study, the entire procedure was completed in a short amount of time with no complications. In histological and immunohistochemical studies, the heart was observed through its outer surface, and the labeled cells and green dye were accurately localized in the LAD area. After 14 days, transplanted cells were found to have engrafted and formed myotubes in normal heart tissue. These results demonstrate that this new direct myocardial injection system allows safe and reliable transplantation of cells into the myocardium and may improve the efficacy of skeletal myoblast transplantation. This new catheter approach could be of clinical significance in the treatment of patients with acute myocardial infarction.
Study limitations. The presence of a branch is indispensable in directing the injection needle when cells are implanted into a target site, thereby limiting its usage to proximal or mid portion of vessels. The maximum diameter of the system restricts its delivery into a distal lesion. Furthermore, this intracoronary delivery system does not allow the catheter to extend beyond the stenosis, unlike our porcine experiment without stenosis. Therefore, the system is suitable only for use with lesions occurring at the initial onset of acute myocardial infarction. Moreover, multiple injections cannot be performed with this device, necessitating intermittent injections to the lesion involved in one branch. Finally, use of the system under two-dimensional angiographic imaging requires expertise in PCI. All these issues are under consideration for further improvement of this novel catheter system.
Acknowledgement. The authors wish to thank Yasuhiro Yanagi of Cardio Inc., Osaka, Japan, for his assistance with this manuscript.
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