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J Thorac Cardiovasc Surg 2001;121:932-942
© 2001 The American Association for Thoracic Surgery
Surgery for Acquired Cardiovascular Disease |
From the Division of Cardiovascular Surgery, Toronto General Hospital, University Health Network, Department of Surgery, University of Toronto, Toronto, Ontario, Canada.
R.K.L. is a Research Scholar of the Heart and Stroke Foundation of Canada. This research was supported by R.K.L.'s research grant from The Hospital for Sick Children Foundation (XG 98-063).
Received for publication May 4, 2000. Revisions requested Oct 25, 2000; revisions received Dec 4, 2000. Accepted for publication Dec 7, 2000. Address for reprints: Ren-Ke Li, MD, PhD, Toronto General Hospital, CCRW 1-815, 101 College St, Toronto, Ontario, Canada M5G 2C4 (E-mail: RenKe.Li{at}UHN.on.ca).
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionAppendix: DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendix: DiscussionReferences |
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1 and may have growth potential. 2 The autologous pericardium, however, is fibrous tissue that has limited elasticity and may shrink, calcify, or dilate and form an aneurysm.
Using cell culture technology, we constructed a cell-seeded biodegradable cardiac graft. 3,4 We seeded cultured rat fetal cardiomyocytes into the gelatin sponge and created 3-dimensional cardiac tissue in a biodegradable graft. Spontaneous contraction occurred when fetal cardiomyocytes were used. 3,4 The beating patch persisted when transplanted into a rat leg or heart, even after the gelatin scaffold dissolved. The beating patch may have the potential to avoid the drawbacks of synthetic materials and autologous pericardium.
The purpose of this study was to assess the feasibility of transplanting a biodegradable cell-seeded patch into the pulmonary circulation. We partially resected the right ventricular outflow tract (RVOT) of syngeneic adult rats and repaired the defect either with a cell-seeded patch or with an unseeded control patch. The patches were examined histologically over the 12-week period.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendix: DiscussionReferences |
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Cell culture and preparation for seeding
Fetal cardiomyocytes and adult heart cells from rat hearts were isolated and cultured by the method we have previously reported. 5 In brief, the hearts were washed in phosphate-buffered saline (PBS) solution (millimoles per liter: NaCl, 136.9; KCl, 2.7; Na2HPO4, 8.1; and KH2PO4, 1.5; pH 7.3). After removal of the atria and the great vessels, the ventricles were minced with fine scissors and incubated in PBS solution containing trypsin (0.25%), collagenase (0.1%), and glucose (0.02%) at 37°C. After 15 minutes' digestion in trypsin and collagenase, the cell suspension was transferred into a tube containing 30 mL of culture medium (Iscove's modified Dulbecco's medium; Gibco Laboratory, Life Technologies, Grand Island, NY) with 10% fetal bovine serum, 0.1 mmol/L of ß-mercaptoethanol, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. The digestion process was repeated 2 times. The cell suspension was centrifuged at 600g for 5 minutes and the cell pellet was resuspended in culture medium. The isolated cells were cultured at 37°C in 5% carbon dioxide and 95% air.
Fetal cardiomyocytes were cultured for 2 days, and the cells were detached from the culture dishes with 1 mL of 0.05% trypsin in PBS solution with glucose (0.02%). After addition of 2 mL of culture medium, the cell suspension was centrifuged at 600g for 5 minutes. The cell pellet was washed, resuspended in culture medium, and centrifuged again. The cell pellet was then resuspended in a minimum amount of culture medium (approximately 50-75 µL) for seeding onto a gelatin sponge patch. The adult heart cells were cultured for 2 weeks. The cells were then treated with trypsin, washed, and resuspended in 50 to 75 µL for seeding onto a gelatin sponge.
Cell seeding onto a gelatin sponge and culture
A sheet of gelatin sponge (Gelfoam: Pharmacia & Upjohn Co, Kalamazoo, Mich) was cut into thin rectangular pieces (15 x 15 x 1 mm) and immersed in culture medium. After the excessive culture medium had been removed from the gelatin sponge by gentle compression, each patch was placed into a 10-cm culture dish. The previously prepared cell pellets were transferred onto the center of each patch (1 x 106 cells/patch) and the patches were incubated. Twenty minutes after incubation, a drop of culture medium was added at each corner of the patch. After another 60 minutes of incubation, 20 mL of culture medium was added to the culture dishes. On the second day after the initial seeding, the patches were turned upside-down in the culture dishes and cell seeding was repeated in the same manner as the initial seeding. After the second seeding, the patches with fetal cardiomyocytes were incubated for 1 week and the adult heart cells were incubated for 3 weeks. Culture medium was changed every 2 days.
Histology of the cell-seeded patch
At the end of the culture period, a sample from fetal or adult cell-seeded patches was fixed by simple immersion in 10% phosphate-buffered formalin solution for 2 days. The fixed patches were cut vertically through the center, and the slices were fixed in 5% glacial acetic acid and methanol, embedded in paraffin, and sectioned to produce 10-µm–thick specimens. The sections were stained with hematoxylin and eosin, as described in the manufacturer's specifications (Sigma Diagnostics, St Louis, Mo).
Cell number and growth curve of the cell-seeded patch
The cells within the patches were counted to generate a growth curve for a period of 1 to 3 weeks (n = 2 for each period). The patch was incubated in 7 mL of PBS solution containing collagenase (0.07%) and glucose (0.02%) at 37°C with agitation until completely dissolved (10-40 minutes). The cell suspension was centrifuged at 600g for 5 minutes, and the cell pellet was resuspended in 1 mL of 0.05% trypsin in PBS solution with glucose (0.02%) at 37°C for 1 minute. Then 9 mL of culture medium was added to terminate digestion. From this cell suspension, 0.25 mL was transferred to 19.75 mL of Isoton II diluent (Coulter Electronics, Inc, Hialeah, Fla), and the number of cells was determined with an automatic cell counter (Coulter Electronics, Inc).
Spontaneous beating of cultured fetal cardiomyocytes in the gelatin sponge
Both the cultured fetal cardiomyocytes and their seeded patches beat spontaneously in the culture dish. Forty-eight hours after plating, the synchronous beating of fetal cell clusters was counted for 1 minute in 5 randomly selected high-power microscopic fields (200x) (n = 10). The pulse rate of the cell-seeded gelatin sponge patch was counted for 1 minute under a low-power microscope (40x) 1 week after seeding (n = 17).
Anesthesia and postoperative care of the recipient rats
Adult male Lewis rats, weighing 250 to 300 g, were anesthetized with an intramuscular injection of ketamine hydrochloride (22 mg/kg), followed by an intraperitoneal injection of sodium pentobarbital (30 mg/kg). The rats were endotracheally intubated and ventilated with a Harvard ventilator (model 683; Harvard Bioscience, Inc, Holliston, Mass). Positive-pressure ventilation was maintained at a rate of 60 cycles/min with a tidal volume of 3 mL and room air supplemented with oxygen (2 L/min). Penlong XL (penicillin G benzathine, 150,000 U/mL, and penicillin G procaine, 150,000 U/mL) was injected intramuscularly (0.4 mL per rat). Buprenorphine hydrochloride (0.01 mg/kg body weight) was given subcutaneously. Postoperatively, rats were monitored until they fully recovered from the anesthesia and were returned to their cages.
RVOT free wall resection and replacement with gelatin sponge patches
The rat heart was exposed via a right lateral thoracotomy under general anesthesia. A purse-string stitch (5-6 mm in a diameter) was placed in the free wall of the RVOT with 7-0 polypropylene sutures (Prolene; Ethicon, Inc, Somerville, NJ). Both ends of the stitch were passed through a 22-gauge plastic vascular cannula (Angiocath; Becton Dickinson and Company, Franklin Lakes, NJ), which was used as a tourniquet. The tourniquet was tightened and the bulging part of the RVOT wall inside the purse-string stitch was resected. The tourniquet was briefly released to determine whether massive bleeding occurred, which indicated that a significant defect had been created in the RVOT. For each type of cell culture, 2 pieces of gelatin sponge patch were laid on top of each other and sewn along the margin of the purse-string suture with over-and-over sutures with 7-0 polypropylene to cover the hole in the RVOT. After completion of suturing, the tourniquet was released and the purse-string stitch was removed.
Additional sutures were added, if needed, to achieve hemostasis. The chest wound was closed in layers with running sutures of 3-0 silk. Ten animals in each group had patches inserted into the RVOT (fetal cardiomyocyte, n = 10; adult cardiomyocyte, n = 10; and unseeded control, n = 10).
Histologic studies
At each scheduled period (4, 8, and 12 weeks), the animals were euthanized (n = 4 at 4 weeks and n = 3 at 8 and 12 weeks in each group) with an intrathoracic injection of 0.2 mL of pentobarbital sodium (Euthanyl; MTC Pharmaceuticals, Cambridge, Ontario, Canada) after injection of 300 units of heparin intramuscularly. Each heart was excised through a repeated median sternotomy and fixed with 10% phosphate-buffered formalin solution for 2 days. The right ventricular free wall was cut in half along the center of the patch. Overall appearance of the endocardial surface was examined. The sections were either stained with hematoxylin and eosin as described above or sent for immunohistochemical staining.
Measurement of the area of the replacement in RVOT
The patch in the RVOT was assessed macroscopically after heart fixation. The endocardial surface of the right ventricular free wall was exposed and photographed (n = 2 in each group). The endocardial area of the patch was measured by computed planimetry (Jandal Scientific Sigma-Scan, Corte Madera, Calif).
Measurement of the thickness of the patch at RVOT
At 4 or 12 weeks after patch implantation, a microscopic image (10x by Labophot microscope; Nikon Corporation, Tokyo, Japan) of the section through the RVOT was taken by camera (FX-35; Nikon Corporation). In each group, 4 rats were examined at 4 weeks and 3 at 12 weeks. A portion of the patch replacing the defect in the RVOT was identified, and the thickness of the center of the patch was measured at the same magnification (10x) by Objective Micrometer (Nikon Corporation). At the same time, 3 normal hearts were harvested and processed in the same manner and the thickness of the RVOT was measured.
Cell identification
The cells expanded in vitro in the gelatin sponge were labeled with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co, St. Louis, Mo). Two days before the RVOT patch implantation procedure, 25 µL of 0.4% BrdU solution was added to the culture dishes of both fetal and adult cardiomyocytes and incubated for 48 hours. A monoclonal antibody against BrdU was used on the heart specimen sections 12 weeks after implantation to localize the labeled cells in the RVOT patches. 6
Assessment of endothelialization with factor VIII staining
A section of the patch in the RVOT was immunohistochemically stained with a monoclonal antibody for the factor VIII–related antigen 7 to assess the endothelialization on the endocardial surface of the patch.
Data analysis
All data are expressed as the mean ± the standard deviation. By means of the SPSS software package for Windows version 9.0 (SPSS Inc, Chicago Ill), the thickness of the RVOT patches in 4 groups, including the control group (n = 3), was compared at 4 weeks and 12 weeks by 1-way analysis of variance. The Scheffé test was used to specify differences between groups. The thickness of the patches at 4 weeks was compared with that at 12 weeks by unpaired t test.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendix: DiscussionReferences |
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Although the cells were seeded on the surface of the gelatin patches, significant cellular infiltration was noted inside the patches (Fig 1). There were no differences in the patterns of cell distribution within the patch for either fetal or adult cardiomyocytes. The structure of the gelatin sponge was maintained for the duration of the in vitro culture.
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, B). The number of cells remained constant after 10 days. From the growth curve, the doubling time was estimated at about 7 days.
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RVOT patch
All animals had a full-thickness replacement of RVOT defect with the gelatin patch (Fig 3). The area of the patch was 8.8 ± 3.0 mm2 (n = 6) at 4 weeks, which was 6.7% ± 2.9% of the endocardial surface area of the free wall of the right ventricle. All RVOT patches became thinner with time (Fig 4) (control group, P = .003; fetal group, P = .001; adult group, P = .07) because of dissolution of the patch. No significant differences were found in the thickness of the patch among the 3 groups, either 4 or 12 weeks after the procedure. However, the patch in the control group was thinner than the normal RVOT (P = .047) at 12 weeks, but the thickness of the fetal and adult cell-seeded patches was similar to that of the normal RVOT.
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, C and D). In the control patches(Fig 5, E and F), the inflammatory reaction was associated with the ingrowth of fibrous tissue from the recipient heart.
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, A). In both heart cell groups, the cells persisted in the patched area. The cells were considered to be the seeded cultured heart cells because they stained positively (brown) for BrdU (Fig 7).
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, B). The endothelialization process started as early as 3 days after the operation. The pattern and the timing of the endothelialization were similar in all the groups. No endocardial thrombus was found in any of the animals of any group put to death 4 or 12 weeks after the operation.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendix: DiscussionReferences |
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8 The group at the Boston Children's Hospital used a biodegradable polymer to create both a pulmonary valve 9 and a pulmonary artery 10 in neonatal lambs. These tissue-engineered grafts persisted after dissolution of the scaffold and grew with the animals. We hope to exploit our ability to grow fetal and neonatal heart cells to create a beating autologous conduit with an autologous valve.
We 3,4 recently reported the construction of a tissue-engineered cardiac graft in vitro, with rat fetal or neonatal cardiomyocytes. A biodegradable gelatin sponge was used as a scaffold to establish cell growth. 11,12 The honeycomb structure of the gelatin sponge allowed the growth of the seeded cells inside the patch, which ensures the adequate perfusion of nutrients and oxygen during culture. Also, the sponge structure was soft enough to permit spontaneous contraction of the fetal or neonatal cardiomyocytes in vitro. In our previous study, we 3 inserted the beating mesh containing either fetal or neonatal cardiomyocytes into the subcutaneous tissue of syngeneic rats, where the patch beat spontaneously for as long as 3 months after the implantation. The seeded cardiomyocytes linked to each other by gap junctions and formed a cardiac tissue, which beat synchronously. Blood vessels were observed in the implanted patch, consisting of an extensive capillary system as well as small arterioles. 3 The beating patch was also attached to the cryoinjured area of the left ventricle. 3 The beating muscle persisted after scaffold dissolution and induced an intense angiogenesis.
Animal model
For this study, we used syngeneic Lewis rats, which avoided rejection, but their small size made our assessment difficult. The RVOT was chosen because a possible application of the tissue-engineered patch would be in patients who require a patch insertion for an RVOT obstruction or stenosis. Additionally, in previously published studies, various patch materials were evaluated in the RVOT of larger animals. 13-15
The major limitation of this small animal model was our difficulty in assessing the hemodynamic effects of the patch in the RVOT. We were unable to determine whether the fetal cardiomyocytes continued to beat synchronously after implantation. We found that the cells formed a tissue, but we could not demonstrate whether the tissue communicated with the host myocardium. Soonpaa and colleagues 16 showed that transplanted fetal cardiomyocytes communicated with the host myocardium by gap junctions. New advances in echocardiography may permit an assessment of regional wall motion of the cell-seeded patches and other hemodynamic information in vivo. 17
Cell-seeded patches
One major challenge to tissue engineering is to induce and maintain cellular growth into the interstices of the biodegradable scaffolding.
In our rat model, the thin patch permitted adequate cell seeding in vitro and adequate perfusion of the graft in vivo. In larger animals and human beings, a thicker patch will be required and adequate seeding and perfusion will be difficult.
To expand cell growth into the interstices of the graft in vitro, we stacked thin cell-seeded grafts. 4 Therefore, we were able to ensure an adequate cell number in the middle of the graft at the time of implantation. The Boston Children's Hospital group also used multiple layers of cell-seeded conduit material to create a new pulmonary artery in lambs. 10 They also used a pulse duplicator to circulate media within their scaffold to create a cell-seeded pulmonary valve. 9 Therefore, techniques to increase cell growth within biodegradable grafts have been successful in larger animals.
In vivo studies demonstrated that cell-seeded grafts induced an intense angiogenic response. 3,4,9,10 In our studies with fetal and neonatal cells in a beating patch transplanted into the leg or onto the left ventricle, we found an extensive capillary network within the beating graft before and after the scaffold dissolved. We also found arterioles and venules in and around the implanted graft. 3 Although many cells did not survive transplantation, our studies and those from Boston 9,10 suggest that angiogenesis permitted survival of a sufficient number of cells to maintain the patch or conduit after the scaffold dissolved.
In the future, we hope to use neonatal heart cells to produce a beating mesh in vitro. We could then implant the autologous beating patch in the RVOT of neonatal animals with the hope that we could create pulsatile pulmonary perfusion. Autologous beating conduits and autologous tissue-engineered valves provide the promise of a complete repair in neonates with complex congenital heart defects.
Patch material
The best biodegradable material for repair of cardiac defects has not been established. Our gelatin sponge is advantageous for creating a new cardiac chamber because it is soft and pliable. However, the gelatin sponge induced an intense inflammatory response. The gelatin sponge is derived from porcine skin protein that may induce a xenogenic reaction. We were surprised that endothelialization developed rapidly on all grafts without evidence of thrombus formation. A more biologically compatible material that does not induce as intense an inflammatory response would be preferable. Improved biomaterials are essential to create a beating patch for human beings.
Future studies may identify a more ideal scaffold for a tissue-engineered cardiac patch. It should be biodegradable, allow for the growth of seeded cells, be soft enough to allow spontaneous beating, and be able to avoid an inflammatory reaction when implanted at a site directly contacting host blood.
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Appendix: Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendix: DiscussionReferences |
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Dr Sakai. We agree that adequate perfusion of the cell-seeded graft is essential. Diffusion may permit support of a thin patch in experimental models, but angiogenesis, which we have noted within 1 to 2 weeks after cell transplantation, may also be required for cell survival in thicker, more clinically useful grafts. This angiogenesis occurs before the gelatin substrate is completely absorbed, which takes more than 5 weeks. We have also found that non–cell-seeded grafts form a fibrous patch after implantation in the right ventricular outflow tract of rats. It may be possible to seed cells into these fibrous patches after implantation in the same way that we have been able to transplant cells into a fibrous myocardial scar.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAppendix: DiscussionReferences |
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P = .036) at 4 weeks, and the control group was thinner (
P = .047) at 12 weeks. FCM, Fetal cardiomyocyte patch; ACM, adult cardiomyocyte patch. *P = .003. **P = .001.
