Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved July 14, 2009 (received for review December 3, 2008)
The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.
The adult heart has limited regeneration capacity, with consequent loss of myocardium and scar formation after extensive myocardial infarction (MI) often leading to heart failure (1, 2). Cell therapy approaches used in recent years, involving the transplantation of suspensions of autologous stem cells or progenitors into the damaged myocardium that have been tested in preclinical and early clinical studies, have only met with marginal success (3, 4). In parallel, cardiac patches produced by tissue engineering methods are being developed for cardiac repair (5–7). Such patches can replace scar tissue or after grafting to the scar, improve cardiac function by supporting and thickening the damaged zone. Another advantage of this approach is that bioengineering of heart muscle can be achieved ex vivo, under precise and controllable conditions.
To realize the therapeutical potential of the bioengineered cardiac patch approach for cardiac repair, vascularization of the patch is a prerequisite for maintaining its viability after transplantation onto the infarcted zone. Ex vivo methods for the prevascularization of skeletal or cardiac muscle patches that involve the coculturing of capillary-forming cells with functional cells have been developed (8, 9). Although the formation of capillaries in ex vivo engineered cardiac patch generated according to such protocols was recently documented (9), the efficacy of such approaches for cardiac repair has not yet been tested. Furthermore, previous work with other patches revealed that in vitro engineered microvessels tended to leak after transplantation, when the comprising endothelial cells had migrated toward the center of the lumen (10).
In the present study, we sought to employ the body as a bioreactor for the engineering of a cardiac patch containing a stable and functional network of blood vessels, before transplantation onto infarcted hearts. Accordingly, we have developed a technique wherein a cardiac patch was first engineered in vitro and then matured and vascularized upon transplantation onto the omentum, a blood vessel-enriched membrane. The patch consisted of neonatal rat heart cells, seeded in macroporous alginate scaffolds designed to allow cardiac cell organization and blood vessel penetration after transplantation (11, 12). To enhance vascularization and patch viability on the omentum, a mixture of prosurvival and angiogenic factors was incorporated into the patch by exploiting their affinity binding to the alginate matrix (13). Seven days after transplantation onto the omentum, the cardiac patch was explanted and transplanted onto 7-day infarcted rat hearts. Cardiac patch engraftment, electrical coupling and cardiac function were evaluated 28 days later by histological, electrophysiological, and echocardiographic examination. The in vivo experiments and analyses were performed by independent technicians and investigators blinded to the experimental groups and the study protocol.
Neonatal rat cardiac cells (2.5 × 106 cells per scaffold) were seeded with factor-reduced Matrigel and a mixture of prosurvival and angiogenic factors into macroporous alginate scaffolds (LVG, 5-mm diameter × 2-mm thick, 100-μm average pore size) (Fig. 1 A–C). The mixture consisted of insulin-like growth factor-1 (IGF-1), stromal-cell derived factor 1 (SDF-1), and VEGF. To achieve controlled presentation and sustained release of the mixture factors, the alginate scaffolds included alginate-sulfate (10% by dry weight), addition of which enables high affinity binding of these factors to the matrix (13). We verified the activity of the mixture factors incorporated into the cardiac patch by demonstrating, via western blot analysis, that the ERK1/2 and Akt signaling pathways, known to promote cardioprotection (14), are extensively activated in these cultures (See Fig. S1A). Using a chemotaxis chamber, we also verified that the release of SDF-1 from the cardiac patch was able to attract human CD34-positive cells to a greater extent, as compared with the attraction elicited by a cardiac patch lacking the supplemented factors (See Fig. S1B).
Construction of a cardiac patch in an alginate-sulfate/alginate scaffold capable of binding and releasing mixture factors. (A and B) The scaffold features before cell seeding; macroscopic view (A) and internal porosity by scanning electron microscopy (B). (C and D) Cardiac patch, seeded with 2.5 × 106 cardiac cells and supplemented with factor mixture, after 48 h of cultivation. (C) Light microscope view of the cardiac patch, showing uniform distribution of cells in the matrix pores. (D) Cardiac cell organization within the scaffold, as judged by anti-actinin immunostaining (green) and nuclear staining (red). Some of the cells reveal the typical striation of cardiac tissue. [Scale bar: 200 μm (C); 10 μm (D).]
The cardiac patch was cultured for 48 h to allow initial tissue organization. Cell distribution throughout the alginate scaffold was confirmed by fluorescein diacetate (FDA)-staining of the patch (See Fig. S1C) whereas the XTT viability assay revealed that the patch retained 100% viable cells. The cardiac cells were organized in clusters within the pores, with some clusters presenting the highly differentiated sarcomeric organization of cardiomyocytes (Fig. 1D). In cardiac patches raised without the supplemented factor mixture, no cardiac muscle structures were seen and cell viability was reduced to 70% of the initial seeded cell number, as shown under static (i.e., with no mixing) cultivation conditions (15).
To induce patch vascularization before grafting onto infarcted heart tissue, we first implanted the mixture-supplemented cardiac patch onto rat omentum for 7 days (n = 8) as we envisioned that the blood vessel-enriched omentum would provide a suitable site for inducing mature vasculature within the patch. To confirm the contribution of factor supplementation to patch vascularization and subsequent cardiac muscle regeneration, patches to which no factors were supplemented were transplanted into control animals (n = 8).
Host blood vessels could be clearly seen entering the mixture-supplemented cardiac patch 7 days after transplantation onto the omentum (Fig. 2A). Hematoxylin and eosin (H&E)-stained tissue cross-sections revealed extensive tissue ingrowth into the mixture-supplemented cardiac patch (Fig. 2B and Fig. S2A for a full cross section of the omental patch) whereas the patches lacking the mixture supplement remained mostly acellular, with most cells present at the patch edges (See Fig. S2B). The tissue within the mixture-supplemented patch was populated with blood vessels, could be immunostained with antibodies to smooth muscle actin (SMA), indicating vessel coverage by pericytes and smooth muscle cells (SMC) (Fig. 2C). The blood vessels were scattered throughout the patch and anastomized to the host vasculature, as judged by their red blood cell content (Fig. 2D). Quantitatively, the vessel density and overall area occupied were 2.5–3 times greater in the mixture-supplemented patches than in the control patches, that is, those patches with no mixture supplementation (Fig. 2 E and F).
Vascularization of the 7 day omentum-transplanted, mixture-supplemented cardiac patch. (A) The cardiac patch (arrow) is stitched to the omentum. No contamination or inflammation were observed in any of the patches (n = 16). (B) H&E-stained cross-section from the omentum-generated cardiac patch supplemented with the prosurvival and angiogenic factors shows extensive tissue in-growth into the scaffold. Lower right is the patch edge. (C) Mature blood vessels populate the cardiac patch supplemented with mixture, as judged by anti-SMA immunostaining (brown). (D) The vessels are functional and anastomized with host vessels, as reflected in their red blood cell content. (E and F) Blood vessel density (E) and the area (F) (in %) occupied by the vessels in the omentum-implanted patches. The results represent mean values ± SEM. (n = 8 per group). Statistical evaluations were performed by unpaired Student's t tests, P < 0.05. (G) Anti-Tn-T immunostaining of thin section in the omentum-generated cardiac patch supplemented with mixture factors (brown). (H) Typical cardiac cell striation is revealed in an omentum-generated, mixture-supplemented cardiac patch, as revealed by anti-actinin immunostaining (green) and confocal microscopy. [Scale bar: 200 μm (B); 100 μm (C); 20 μm (D and G); 10 μm (H).]
We then assessed cardiac muscle formation in the omentum-generated patches by immunostaining with antibodies against cardiac muscle markers, such as troponin T (Tn-T) and actinin. In the mixture-supplemented cardiac patches, bundles of cardiac muscle structures could be identified throughout the entire patch, as revealed by their distinctive Tn-T-staining (Fig. 2G). The cardiac muscle structures revealed typical cardiac cell striation, indicating cardiac muscle formation with those patches fused onto the omentum (Fig. 2H, green reflects anti-actinin antibody immunostaining). By contrast, in those cardiac patches where no mixture supplementation had been made, no distinctive cardiac muscle structures were observed 7 days after attachment to the omentum (See Fig. S2 C and D).
The prevascularized cardiac patches were implanted onto the infarcted heart of male SD rats (n = 11) 7 days after MI, induced by ligation of the left descending coronary artery (See Fig. S3). Stitched rat hearts after MI induction served as negative controls (n = 7) whereas the implantation of in vitro-grown mixture-supplemented cardiac patches (n = 6) served to test for the effects of prevascularization on patch grafting onto infarcted myocardium and subsequent cardiac repair. Only rats displaying fractional shortening (FS) < 40% as assessed by echocardiography were included in study.
Four weeks after implantation, the omentum-generated patches were fully integrated into the host myocardium and showed thicker scars than those observed on the myocardium of control rats (Fig. 3 A–C). The prevascularized patch was populated with striated and elongated cardiac cells, some of which were oriented perpendicular to the direction of the host myocardium (Fig. 3 C and D). The cardiac muscle structures could be positively stained with antibodies to Tn-T (Fig. 3E) and connexin 43 (Cx-43) (Fig. 3F), indicating the formation of mechanical contacts between the transplanted cells. By contrast, in most of the animals into which the in vitro grown cardiac patches were transplanted, processing for histological examination led to the patches being detached from the host myocardium, indicative of these patches not integrating with the myocardial scar (Fig. 3B).
Assessment of the scar zone, 28 days after patch grafting onto an infarcted heart. (A–C) Representative figures of Masson's trichrome-stained cross-sections in a stitched only scar (A), a scar implanted with an in vitro-grown patch (B), or a scar grafted with an omentum-generated cardiac patch (C). Collagen in the scar is stained blue and viable cardiac tissue is shown in red. (D) H&E staining of cross-sections in the interface (dashed black line) of the host myocardium (M) and grafted omentum-generated patch (P). (E) Typical cardiac cell striation could be observed by anti-Tn-T immunostaining (brown). (F) Cx-43 expression (brown) between adjacent cardiomyocytes in omentum-generated cardiac patch suggests mechanical coupling. (G and H) Morphometric analysis of scar area and calculation of relative scar thickness (G) and expansion index (H), [(LV cavity area/whole LV area)/relative scar thickness]. (I) Blood vessel density in the scar area was determined by counting anti-SMA-immunostained vessels. Results represent mean values ± SEM. (n = 4–6). Statistical evaluations were performed by unpaired Student's t tests, P < 0.05. [Scale bar: 500 μm (A–C); 200 μm (D); 20 μm (E and F).]
Morphometric analysis showed that the relative scar thickness (average scar thickness/average wall thickness) was significantly greater in the omentum-generated patch-treated hearts, with a value close to 1, reflecting normal wall thickness (Fig. 3G). Moreover, the infarct expansion index [(LV cavity area/whole LV area)/relative scar thickness] was much smaller in the omentum-generated patch-treated hearts than in hearts of the control groups (Fig. 3H). Together, these data indicate that transplantation of the omentum-generated cardiac patch is attenuating LV dilatation after MI.
When evaluating blood vessel density in scars treated with omentum-generated patches (Fig. 3I), the density measured (65±9/mm2) is close to that found in the patches before their implantation onto the infarct (48±4/mm2, Fig. 2E), possibly indicating that the scar neovessels mainly originated from the integrated patch. In infarcts treated with the in vitro-grown cardiac patch, vessel density was similar to that observed in the stitched scars, consistent with the lack of patch integration into the scar in these cases.
Electrical coupling of the omentum-generated cardiac patch to the host myocardium was assessed 4 weeks after engraftment using Langendorff-perfused isolated heart preparations (n = 3). Similar preparations from empty scaffold-treated (n = 2, to rule out biomaterial effect) and stitched (n = 2) infarcted hearts served as controls. To assess the level of electrical connectivity between the engrafted patch and the myocardial tissue, two miniature bipolar hook electrodes were blindly inserted on the epicardium at the base of the right ventricle (RV, healthy myocardium electrode) and on the scar zone, as close as possible to the stitch (scar electrode). As expected, spontaneous electrical activity was greater in healthy myocardium, as compared with scar tissue, in all seven preparations. Recordings from scars treated with omentum-generated cardiac patches showed greater amplitude signals than did recordings made from the scars of control subjects (3.32 ± 0.32 mV vs. 1.55 ± 0.34 mV, respectively) (Fig. 4 A, B, and E). Consistent with this observation, pacing through the scar electrode (2 ms square pulses, 400 beats per minute) resulted in a much lower scar capture threshold (defined as the minimal stimulus intensity needed to reach synchronous activation of the healthy tissue by pacing through the scar electrode) in hearts treated with the omentum-generated patches than was the case with stitched scars (1.16 ± 0.16 mA vs. 5.5 ± 0.5 mA, respectively), indicating improved electrical connectivity between the scar zone and the healthy myocardium in those preparations treated with omentum-generated patches (Fig. 4 C, D, and F).
Electrical coupling of omentum-generated cardiac patch to host myocardium. Langendorff-perfused isolated hearts were blindly implanted with two bipolar epicardial electrodes located at the base of the healthy right ventricle myocardium (RV) and the scar zone as close as possible to the stitch (scar). (A and B) Nonpaced (spontaneous) electrical signals recorded from the healthy myocardium (RV) and scar zone (Scar) of hearts with an omentum-generated patch (A) or hearts with stitch only (B). (C and D) Pacing through the scar electrode using 2 ms square pulses (400 beats per min) in hearts treated with an omentum-generated patch (C) or stitched scar (D), at a stimulus intensity of 1.5 mA. This stimulus intensity could capture all hearts treated with omentum-generated patch, but none of the control hearts. (E) Comparison of signal amplitude in the scar zone after grafting an omentum-generated patch (black) or in stitched hearts (white). (F) Comparison of capture threshold intensity in hearts grafted with an omentum-generated patch (black) or only stitched (white).
Two-dimensional (2D) echocardiographic examinations were performed 6 days after MI induction, to obtain baseline readings. Thirty of the 37 studied rats showed fractional shortening (FS) <40%; these were subsequently subjected to echocardiography study, 28 days after patch grafting or scar stitching. Herein, an additional group of implanted a-cellular constructs supplemented with the factor mixture and grown on the omentum for 7 days (n = 6) was transplanted on the infarcted hearts, to assess the omentum contribution to cardiac function.
Evaluation of the baseline showed the formation of scar tissue 6 days after MI induction. Twenty-eight days after intervention, the fractional area change (FAC) and fractional shortening (FS) echocardiography data indicated further deterioration in the stitched infarcts and in hearts implanted with in grown patches (Fig. 5 A and B, Fig. S4 A and B, and Table S1). By contrast, FAC and FS values did not differ from baseline values in animals treated with the omentum-generated patches with the cellular patches being more invariant (cardiac patches: P = 0.22 and 0.6; a-cellular patches: P = 0.09 and 0.17, in terms of percent change in FAC and FS, respectively) (Fig. 5 C and D and Fig. S4 C and D). Additionally, the percent changes in FAC, LV end diastolic dimension (LVEDD) and LV end systolic dimension (LVESD) were all significantly smaller in animals treated with the omentum-generated cardiac patches compared with the animals with the stitched hearts (Fig. 5 E–G and Table S2).
Changes in left ventricle function after patch grafting. The FAC {[(LV end-diastolic area − LV end-systolic area)/LV end-diastolic area] ×100} of infarcted hearts treated with stitches only (A) (n = 7), hearts treated with in vitro-grown patches (B) (n = 6), treated with a-cellular omentum-grown scaffolds (C) (Om., n = 6), or treated with omentum-generated cardiac patches (D) (Om.+, n = 11), was determined by echocardiography. “Pre” indicates readings 6 days after MI induction by LAD ligation and 1 day before intervention. “Post” indicates readings taken 28 days after intervention. Comparison of FAC (E) change, LVEDD (F), and LVESD (G). Changes were calculated as follows: [(values obtained after 4 weeks – baseline values)/baseline values] × 100%. Statistical evaluations were performed by paired Student's t tests, P < 0.05 (A–D) or un-paired t test and one-way ANOVA, P < 0.05 (E–G).
This study demonstrates that the omentum can serve as an efficient bioreactor for producing cardiac patches with functional blood vessel network, which, after successful grafting to infarcts, attenuate LV remodeling and dysfunction in rats that experienced MI. The functional vascular network that developed in omentum-generated cardiac patches enabled the maintenance of transplanted and resident viable cardiac muscle structures. After grafting onto scar tissue, the cardiac patches were structurally and electrically coupled with the host myocardium, leading to beneficial effects on systolic and diastolic left ventricular function.
A prerequisite for successful myocardial regeneration is that the implanted cardiac patch retains viability and integrates into the infarct. Prevascularization of the patch and rapid anastemosis of the patch vasculature with host myocardial vessels would ensure the rapid engraftment and integration of the patch into the infarcted myocardium. Implantation on omentum for inducing vasculogenesis in tissue explants was previously explored (16–18). Here, we have used the omentum for prevascularization and cardiac tissue engineering given that this tissue is enriched with blood vessels.
The incorporation of prosurvival and angiogenic factors into the cardiac patch protected the scaffold-seeded cells and induced cardiac muscle formation during in vitro cultivation and after patch implantation, increased survival of the nascent cardiac tissue and induced cardiac tissue maturation on the omentum. Furthermore, the mixture factors enhanced patch vascularization on the omentum, inducing the formation of a stable network of functional blood vessels to nourish the transplanted cardiac tissue within the patch. The mixture consisted of Matrigel, shown to prevent anoikis (19) and enhance capillary formation (20), SDF-1, a chemoattractant for bone marrow-derived stem cells (BMSC) and endothelial progenitor cells (EPC) (21, 22), and shown to act as a cardioprotective agent (23), IGF-1, a cardioprotective and angiogenic agent (24), and VEGF, an angiogenic factor (12, 25, 26). The maintenance of mixture factor activity in the patch during in vitro cultivation and later, in vivo, is attributed to their affinity binding to alginate-sulfate within the matrix and via their sustained release (13, 27).
An important observation of this study is that the omentum-generated prevascularized patch was structurally and electrically integrated into the host myocardium, 28 days after transplantation onto the infarct. By this point, the scaffold disappeared from the infarct, thereby allowing the bioengineered cardiac tissue to form mechanical and electrical contacts with the host myocardium. The successful grafting of the omentum-generated patch resulted in the formation of thicker scars with an average relative scar thickness close to 1, the same value as measured for the normal LV wall and 2-fold higher than in the control groups. Importantly, the electrical recordings provided compelling evidence for the improved electrical coupling between the omentum-generated patch and healthy myocardium, as evidenced by the higher amplitude of electrical signals in the scar zone and by the markedly lower capture threshold for pacing, indicating better excitability and/or electrical connectivity between the scar and healthy myocardium.
The integration of the omentum-generated cardiac patch to the host myocardium is attributed to the fact that bioengineering on the omentum resulted in a consistent tissue with a stable network of blood vessels able to anastomize with the host myocardium. Consistent with this claim is the finding that blood vessel density in the transplanted omentum-generated patch resembled that of the scar zone after grafting. Further, infiltration of host cells like fibroblasts possibly contributed to integration by enhancing patch adhesion to the scar. By contrast, the in vitro grown patch, although initially supplemented with the same mixture factors, could not integrate into the infarct because of the acellular nature of patches so raised.
The most important outcome of the successful grafting of the omentum-generated cardiac patch to scar tissue was its ability to improve after MI cardiac function (% change in FS) and to inhibit ventricular dilatation (% change in FAC) after MI. The effect of treatment on these parameters was more pronounced in animals implanted with omentum-generated cardiac cellular patches than in those transplanted with omentum-generated a-cellular patches (although the difference between these groups did not reach significance). We attribute this to the addition via patch grafting of contractile structures to the infarct site as seen in Fig. 3 C–F. Additionally, the cellular and acellular omentum-generated patches could contribute to enhanced angiogenesis and to preservation of remaining resident cardiac tissue at the infarct zone after MI; the two patches affinity-bound and deliver angiogenic and cardioprotective factors as well as their generation on the omentum most likely supplement them with additional cells and factors from the omentum which add to their beneficial effects.
The successful cardiac patch grafting onto the scar increased scar thickness and stabilized the chamber size. By thickening the scar, wall stress is reduced (according to Laplace's law) and the degree of outward motion of the infarct that occurs during systole (dyskinesis) is reduced. This effect is significant because one of the most important predictors of mortality in patients after MI is the degree of LV systolic dilatation (28). During the echocardiography studies, we observed variability in infarct sizes and functional consequences, in those rats with initially large infarcts (FS, 10–30%) showing substantial improvement after treatment with engineered cardiac tissues. Our results are in agreement with those of Zimmermann et al. (29), indicating that the cardiac patch approach is able to treat large infarcts and improve cardiac function after MI.
Using the body as a bioreactor to engineer cardiac tissue with stable and functional blood vessel networks represents a significant improvement in cardiac patch performance over ex vivo methods currently used for patch production. Others have successfully exploited the omentum for the vascularization of hepatic tissues and for bladder construction (30, 31). Recently, our group transplanted cardiac cell-seeded scaffolds into the peritoneal cavity in an attempt to induce tissue engineering of the patch (32). In those studies, however, no cardiac muscle structures were detected in the peritoneal-generated patch, with tissue in-growth consisting of myofibroblasts being embedded into collagen bundles and infiltrated with blood vessels. The results of the present study reveal that cardiac muscle structures are regenerated on the omentum and can survive transplantation onto infarcted heart in the presence of an appropriate mature vasculature. Furthermore, our study reports structural and electrical integration of a cardiac patch resulting in improved after MI heart function. This can be attributed to the improved prevascularization of the cardiac patch by supplementation of mixture factors.
We realize that explanting the cardiac patch from the omentum is associated with transient discontinuation of blood flow to engineered tissue. Yet, the time interval from explantation to implantation on the infarct is short (<5 min) and the vessel infrastructure is probably preserved during this time interval. It is also likely that blood fluid remaining in the graft vessels throughout this procedure, nourishes the cells until anastomosis with the host is achieved. The survival of the engineered cardiac tissue after explantation and implantation on the infarct and the therapeutic outcome of the patch support the continuous supply of oxygen and nutrients to the cells.
Surgically, the procedure described herein can be applied in human because of its simplicity, safety and efficacy for cardiac repair. However, because most MI patients are old and multiple surgeries can pose a large risk on them, the clinical application of our strategy is currently not an option for these patients. Regenerative strategies by employing instructive biomaterials or combination of biomaterials and growth factors should be further explored as more simple approaches for cardiac regeneration after MI (5).
The study, performed in accordance with the guidelines of the Animal Care and Use Committee of Sheba Medical Center, Tel Aviv University, was approved by the institutional review board and was supervised by institutional animal protection officials.
The cardiac patch was prepared as described (15, 33). Briefly, cardiac cells were isolated from the LV of SD neonatal (1–2 days old) rat hearts and seeded onto alginate-sulfate/alginate scaffolds (5 × 2-mm, d × h, 0.7 × 108 cells per cm3). In cardiac patches supplemented with mixture, the cells were seeded with Matrigel (30% vol/vol) and SDF-1, IGF-1, and VEGF (100 ng each) in M199 medium. After cultivation under static conditions in a humidified incubator for 48 h, the patches were analyzed for viability or implanted. All factors, excluding the cells were added to the a-cellular patches.
Recipient SD male rats (150–200 g) were anesthetized with a combination of ketamine (40 mg/kg) and xylazine (10 mg/kg). After a midline abdominal incision was made, the patches (after 48 h cultivation) were placed on the omentum and were secured in place with a 6–0 prolene suture. Seven days after transplantation, the animals were killed and the patches were gently cut and cleaned with scalpel. The patches were then either taken for evaluation or transplanted onto the infarcted heart of a rat MI model.
MI was induced as described in ref. 6. SD male rats (≈250 g) were anesthetized with a combination of ketamine (40 mg/kg) and xylazine (10 mg/kg), intubated, and mechanically ventilated. The chest was open by left thoracotomy, the pericardium was removed, and the proximal left coronary artery was permanently occulated with an intramural stitch. Seven days after MI, omentum-generated patches (acellular or containing cardiac cells) or in vitro-grown (7 days under static conditions) patches were implanted onto the scar tissue by a single stitch. Twenty-eight days after implantation, the rats were either killed or taken for further evaluation.
For morphometric analyses, the slides were stained with Masson's trichrome, microscopically examined and analyzed with Cell-P software (Olympus). The average LV wall thickness was calculated from three measurements of septum thickness whereas average scar thickness was determined from three measurements of scar thickness in each animal. Relative scar thickness was calculated as average scar thickness divided by average wall thickness. The expansion index was calculated as follows: expansion index = [(LV cavity area/whole LV area)/relative scar thickness].
Hearts were excised as described in ref. 34. Briefly, rats were heparinized (500 U/kg, IP) and hearts were rapidly excised with constant perfusion with Hepes Tyrode's solution preheated to 37 °C (100-mm perfusion pressure). Miniature bipolar hook electrodes were attached to the epicardial surface at the patch/scar and to the base portion of the right ventricle (34). Electrophysiological signals were recorded, filtered and interfaced with a PC using an A/D converter (PCI-6024E, National Instruments) and a program developed by YE, as described in ref. 34. Electrical stimulation (2 ms square pulses) was applied through an optically isolated pacer (Pulsar 6bp-as, FHC). The operator of these experiments was blinded as to whether the examined animal was treated with the omentum-generated patch (n = 3), by stitch only (n = 2) or with an empty scaffold (n = 2).
Transthoracic echocardiography was performed on all animals, 6 days after MI (baseline echocardiogram) and 28 days after intervention. Echocardiograms were performed with commercially available echocardiography system (Vivid i, GE Healthcare) equipped with a 12-MHz phased-array transducer (Hewlett Packard). All measurements were averaged over three consecutive cardiac cycles and were performed by an experienced technician blinded to the treatment group.
Statistical analysis data are presented as means ± SEM. Univariate differences between the control and treated groups were assessed with Student's t test. Changes in echocardiographic data between baseline readings and 28 days after treatment, as well as LV function, were assessed by paired and unpaired t tests and by one-way ANOVA. All analyses were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software). P < 0.05 was considered significant.
For additional information see SI Text.
We thank Ms. Lena Shoval for technical assistance. This work was supported by Israel Science Foundation Grant 793/04. S.C. holds the Claire and Harold Oshry Professor Chair in Biotechnology. The work is part of the doctoral thesis of T.D. at Ben-Gurion University of the Negev.
Author contributions: T.D., J.L., and S.C. designed research; T.D., A.K., E.R., O.L., N.L., R.H., and S.D. performed research; I.F. and Y.E. contributed new reagents/analytic tools; T.D., E.R., M.S.F., Y.E., J.L., and S.C. analyzed data; and T.D. and S.C. wrote the paper.
Conflict of interest statement: Y.E. and Mor Research Applications Ltd. have applied for a patent on the miniature bipolar hook electrode (International Patent Application No. PCT/IL2008/000161).
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0812242106/DCSupplemental.
