Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and induces neovasculature formation in ischemic myocardium
Karen L. Christman, BS*,
Andrew J. Vardanian, MS ,
Qizhi Fang, MD ,
Richard E. Sievers, BS,
Hubert H. Fok, MD and
Randall J. Lee, MD, PhD*,*
* University of California Berkeley and San Francisco Joint Bioengineering Graduate Group, University of California San Francisco, San Francisco, California, USA
Department of Medicine, University of California San Francisco, San Francisco, California, USA
Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, USA
Manuscript received December 2, 2003;
revised manuscript received March 22, 2004,
accepted April 13, 2004.
* Reprint requests and correspondence: Dr. Randall J. Lee, Cardiac Electrophysiology, MU East Tower, Box 1354, 500 Parnassus Avenue, San Francisco, California 94143-1354, USA.
lee{at}medicine.ucsf.edu
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Abstract
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OBJECTIVES: In this study, we determined whether fibrin glue improves cell transplant retention and survival, reduces infarct expansion, and induces neovasculature formation.
BACKGROUND: Current efforts in restoring the myocardium after myocardial infarction (MI) include the delivery of viable cells to replace necrotic cardiomyocytes. Cellular transplantation techniques are, however, limited by transplanted cell retention and survival within the ischemic tissue.
METHODS: The left coronary artery of rats was occluded for 17 min followed by reperfusion. One week later, bovine serum albumin (BSA), fibrin glue, skeletal myoblasts in BSA, or skeletal myoblasts in fibrin glue were injected into the infarcted area of the left ventricle. The animals were euthanized five weeks after injection, and their hearts were excised, fresh frozen, and sectioned for histology and immunohistochemistry.
RESULTS: After five weeks, the mean area covered by skeletal myoblasts in fibrin glue was significantly greater than the area covered by myoblasts injected in BSA. Myoblasts within the infarct were often concentrated around arterioles. The infarct scar size and myoblasts in the fibrin group were significantly smaller than those in the control and BSA groups. Fibrin glue also significantly increased the arteriole density in the infarct scar as compared with the control group.
CONCLUSIONS: This study indicates that fibrin glue increases cell transplant survival, decreases infarct size, and increases blood flow to ischemic myocardium. Therefore, fibrin glue may have potential as a biomaterial scaffold to improve cellular cardiomyoplasty treat and MIs.
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Abbreviations and Acronyms
| | BSA | = bovine serum albumin | | DAPI | = 4',6-diamidino-2-phenylindole | | LV | = left ventricle | | MHC | = myosin heavy chain | | MI | = myocardial infarction | | PBS | = phosphate-buffered saline |
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Heart failure following a myocardial infarction (MI) is often progressive. Scar tissue formation and aneurysmal thinning of the infarct region often occur in patients who survive MIs, leading to congestive heart failure. Currently, heart transplantation is the only successful treatment for end-stage heart failure; however, the ability to provide this treatment is limited by the availability of donor hearts.
Cellular cardiomyoplasty involves the transplantation of viable cells to replace the necrotic cardiomyocytes. Several groups have examined delivering a variety of cell types, including skeletal myoblasts, fetal and neonatal cardiomyocytes, and embryonic and adult stem cells to the ischemic myocardium (1–11). The current transplantation technique involves the injection of cells afloat in saline, cell culture medium, or bovine serum albumin (BSA) and results in viable grafts; however, the technique is plagued by limited cell retention and transplant survival (5,8,11,12). It was recently stated that the "basic protocol for cell grafting may need further optimization to prevent cell loss" (12).
The emerging field of tissue engineering may provide promising alternatives. Tissue-engineering approaches are designed to repair lost or damaged tissue through the use of cellular transplantation and biomaterial scaffolds. Other groups have used this tissue-engineering approach by implanting cells on the surface of the myocardium in a polymer scaffold (13,14); however, this approach does not deliver viable cells into the infarct wall. Moreover, there is a limit on the thickness of these cultured implants due to lack of vascularization.
In this study, we examine a novel approach to heart repair that uses an injectable biopolymeric scaffold to deliver cells directly into the infarct wall. We hypothesized that the injection of cells in a solution that becomes a semi-rigid scaffold upon injection would increase cell transplant retention and survival within the infarct, compared with the standard injection technique. The bioactive fibrin scaffold is also known to be angiogenic (15–18), and we examined whether this material could promote angiogenesis and arteriogenesis in ischemic myocardium. We have previously reported that the injection of fibrin glue preserves cardiac function and wall thickness after acute ischemic myocardial injury (19). In this study, we further examined fibrin glue's effect on left ventricle (LV) remodeling by examining its effect on infarct size. These hypotheses were tested using a rat acute MI and allograft transplantation model.
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Methods
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This study was approved by the Committee for Animal Research of the University of California San Francisco and was performed in accordance with the recommendations of the American Association for Accreditation of Laboratory Animal Care (Rockville, Maryland).
Rat MI model.
A previously described ischemia reperfusion model was used in this study (20). Briefly, female Sprague-Dawley rats (225 to 250 g) were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg). Under sterile technique, the rats were placed in a supine position and the chests were cleaned and shaved. The chests were then opened by performing a median sternotomy, and the pericardial sacs were removed. While the landmarks of the base of the left atrium and the interventricular groove were kept in view, a single stitch of 7-O Ticron (U.S. Surgical, Norwalk, Connecticut) suture was placed through the myocardium at a depth slightly greater than the perceived level of the left coronary artery, taking care not to enter the ventricular chamber. The suture was tightened to occlude the artery for 17 min and then removed to allow for reperfusion. The chest was then closed, and the animal was allowed to recover for one week. This laboratory has extensive experience with this model and has previously demonstrated that this technique results in an acute infarct size of approximately 30% of the LV with reperfusion (21–23).
Skeletal myoblast isolation and culture.
Myoblasts from the hind limb muscle of one litter of Sprague-Dawley neonatal rats (10 to 12 rats; 2 to 5 days old) were isolated and purified according to the following described procedure (24). Briefly, the hind limb was harvested under phosphate-buffered saline (PBS)-penicillin/streptomycin and mechanically minced. The tissue was enzymatically dissociated with dispase and collagenase (Worthington, Lakewood, New Jersey) in Dulbecco's PBS (Sigma Chemical Co., St. Louis, Missouri) for 45 min at 37°C. The resulting suspension was then passed through an 80-µm filter, and the cells that passed through the filter were collected by centrifugation. The cells were preplated for 10 min to isolate myoblasts from fibroblasts since fibroblasts plate quicker than myoblasts. The myoblast suspension was transferred to a collagen-coated 100-mm polystyrene tissue culture dish and allowed to proliferate in growth media (80% Ham's F10C media, 20% fetal bovine serum, 1% penicillin/streptomycin, 2.5 ng/ml recombinant human basic fibroblast growth factor) at 37°C in a humidified atmosphere of 95% air plus 5% CO2. Cultures were allowed to reach a confluency of 70% to 75% and passaged every three to four days (1:4 split). Cultures were examined for fibroblast contamination, and only populations of >95% myoblasts were acceptable for injection. To verify the percentage of myoblasts in the population, cultured cells were stained with desmin (Sigma; 1:20 dilution) to label myoblasts and Hoechst 33342 (Molecular Probes, Eugene, Oregon) to label nuclei. Rat fibroblasts (American Type Culture Collection, Rockville, Minnesota) and L6 rat myoblasts (American Type Culture Collection) were also stained as negative and positive controls, respectively (Fig. 1). All injections were from the same pool of cells. Prior to injecting the rats, which were sacrificed 24 h after injection, the myoblasts were labeled with 4',6-diamidino-2-phenylindole (DAPI) for 25 min (3 µM; Molecular Probes).
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Figure 1 Transplant myoblasts. (A) Phase contrast images. (B) Desmin-labeled cells. (C) Hoechst 33342-labeled nuclei. Transplant rat myoblasts (bottom panel) were stained with desmin and hoechst to verify myoblast percentage. Desmin staining indicates that cultured cell populations, which were subsequently injected into the myocardium, contained >95% myoblasts. One contaminating fibroblast is indicated by the arrows. Control rat fibroblasts and rat myoblasts from the L6 cell line were stained with desmin and hoechst as negative and positive controls, respectively.
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Fibrin glue.
The fibrin glue used in this study was the commercially available Tisseel VH fibrin sealant (Baxter Healthcare Corp., Glendale, California). It is a two-component system that remains liquid for several seconds before solidifying into a solid gel matrix. The first component consists of concentrated fibrinogen and aprotinin, a fibrinolysis inhibitor. The second is a mixture of thrombin and CaCl2. It is delivered through the supplied Duploject applicator, which holds the two components in separate syringes and provides simultaneous mixing and delivery. The ratio of fibrinogen to thrombin components was 1:1.
In vitro cell survival in fibrin glue.
To determine whether skeletal myoblasts were capable of surviving in fibrin glue, 75 µl of the thrombin solution containing 2 x 105 myoblasts per microliter was combined with 75 µl of fibrinogen solution and plated onto four-well chamber slides (1.7-cm2) dishes. Growth media were added to the dishes and changed every three days. At one day, three days, and seven days in culture, a Live/Dead stain (Molecular Probes) was used to label live and dead cells. Each culture time point was performed in duplicate. Five microliters of 2 mM ethidium homodimer-1 solution and 1.25 µl of 4 mM calcein was added to 10 ml of PBS. Each chamber was incubated with 200 µl of this solution for 30 min. The myoblast-fibrin glue cultures were then rinsed with PBS and examined using a Nikon TE 300 fluorescent microscope (Nikon, Melville, New York) for the presence of both surviving and non-surviving cells.
Injection surgeries.
One week after MI, either 0.5% BSA in 50 µl of PBS, 50 µl of fibrin glue, 5 x 106 myoblasts in 50 µl of 0.5% BSA, or 5 x 106 myoblasts in 50 µl of fibrin glue was injected into the infarcted myocardium. Under sterile technique, the rats were anesthetized, and the abdomens were opened from the xiphoid process to a left subaxillar level along the lower rib. The LV apex was exposed via a subdiaphragmatic incision, leaving the chest wall and sternum intact. Rats were randomized to either control or treatment groups, and injections were made through a 30-gauge needle into the infarcted area of the LV. The ischemic area was identified by a darker region of the LV that has limited contractility. Injections were made at an angle to reduce the likelihood of injecting into the lumen of the LV. Injections were verified by a slight lightening in color of the myocardium as the solutions entered the infarct wall. Injections were successful in each animal. One injection with a volume of 50 µl was performed on each animal. In the cells group, 5 x 106 myoblasts were suspended in 50 µl of 0.5% BSA and injected into the myocardium. In the cells in fibrin group, 5 x 106 myoblasts were suspended in 25 µl of the thrombin component of the fibrin glue. The thrombin-cell mixture was simultaneously injected into the myocardium with 25 µl of the fibrinogen component via the Baxter's supplied Duploject applicator, which provides simultaneous mixing and delivery. Twenty-five microliters of thrombin and 25 µl of fibrinogen were simultaneously injected into ischemic myocardium in the fibrin group. The diaphragm was sutured closed after suction of the chest cavity, and the abdomen was subsequently closed.
Histology.
Either 24 h or 5 weeks after the injection surgeries, the rats were euthanized with a pentobarbital overdose (200 mg/kg). The study was concluded six weeks after infarction, at which point the remodeling process in rats is complete (25). The hearts were rapidly excised and fresh frozen in Tissue Tek O.C.T. freezing medium (Sakura, Torrance, California). They were then sectioned into 5-µm slices and stained with hematoxylin and eosin. Five slides, equally distributed through the infarct area, were taken from each heart as a representative sample and measured for infarct size as previously described (26). Briefly, the infarct and LV were traced, and size was determined using planimetry. Infarct scar size was determined as the infarct scar area divided by the total LV area as measured with SPOT 3.5.1 software (Diagnostic Instruments, Sterling Heights, Michigan) and recorded as a percentage of the LV. Five additional slides from both the 24-h cells in BSA group (n = 5) and 24-h cells in fibrin group (n = 4) were examined for presence of DAPI-labeled transplanted cells. To measure cell retention within the myocardium, the area covered by the DAPI-labeled myoblasts was traced using SPOT 3.5.1 (Diagnostic Instrument Inc., Sterling Heights, Michigan) according to a previously described procedure, which used an orange cell tracker rather than DAPI (11), and was expressed as percentage of the infarct area. All hematoxylin and eosin stained slides also were examined for any evidence of an immune reaction by our cardiac pathologist.
Immunohistochemistry.
Five slides, equally distributed through the infarct area, were also taken from each heart in the five-week BSA group (n = 6), five-week fibrin group (n = 5), five-week myoblasts in BSA group (n = 5), and five-week myoblast in fibrin group and stained with an anti-smooth muscle actin antibody (Dako; 1:75 dilution) to label microvessels that are predominately arterioles (27). Five slides also were taken from each heart in the five-week myoblasts group (n = 5) and five-week myoblasts in fibrin group (n = 5) after sacrifice and stained with the MY-32 clone (Sigma; 1:400 dilution), which is directed against the skeletal fast isoform of myosin heavy chain (MHC) (28), in order to label transplanted cells. Sections of rat hind limb skeletal muscle also were stained with the anti-skeletal MHC antibody to serve as a positive control. Sections that were incubated only with the secondary antibody were used as negative controls. Slides were initially fixed in 1.5% formaldehyde and then blocked with staining buffer (0.3% Triton X-100 and 2% normal goat serum in PBS). Sections were incubated with the primary antibody diluted in staining buffer. In order to visualize labeled arterioles and skeletal myoblasts, sections were incubated with a Cy-3-conjugated anti-mouse secondary antibody (Sigma; 1:100 dilution). Sections were mounted with Gel/Mount (Biomeda). Alpha-smooth muscle actin labeled microvessels in each section were quantified using the following criteria: 1) positive for smooth muscle labeling; 2) within or bordering the infarct scar; 3) having a visible lumen; and 4) having a diameter between 10 and 100 µm. The scar area was measured using SPOT 3.5.1 software, and arteriole densities were calculated. Cell survival was determined according to a previously described procedure (29) by measuring the area covered by cells that stained positive for anti-skeletal fast MHC using Scion Image (Scion, Frederick, Maryland). Cell area was reported as percentage of infarct area. This method of determining cell survival addresses the amount of transplant cells surviving in the myocardium and does not necessarily determine those transplant cells that have undergone engraftment. Five additional slides were taken from each heart in all of the five-week groups. Capillaries were labeled using a previously described procedure (14). Slides were fixed in room-temperature acetone, and endogenous peroxide activity was quenched with 3% H2O2. Sections were incubated with biotinylated Griffonia simplicifolia lectin (GSL-1; Vector Labs; Burlingame, California). Sections were then incubated with peroxidase-conjugated streptavidin (LSAB2 System, HRP, DakoCytomation, Glostrup, Denmark), capillaries were visualized using 3,3'-diaminobenzidine chromagen solution (LSAB2 System), and sections were mounted with Gel/Mount. Five high-magnification fields within the infarct of each section were chosen at random, capillaries were counted, and vessel density was calculated.
Statistical analysis.
Data are presented as mean ± standard deviation. Cell density measurements were compared using the student t test. Infarct size and vessel measurements were compared using one-way analysis of variance with Holm's adjustment. Significance was accepted at p < 0.05.
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Results
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A total of 43 rats were used in this study. Eight rats died during or immediately after the infarct surgery, whereas one rat died during the injection surgery (cells in fibrin glue group). After the injection surgery, there was 100% survival in all groups.
In vitro cell survival in fibrin glue.
Myoblasts were capable of surviving and proliferating in fibrin glue up to seven days in culture. There was little, if any, cell death caused by the matrix (Fig. 2). Myoblasts had greatly proliferated and were at a high density within the matrix after culture for seven days in growth media.
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Figure 2 Myoblast survival in fibrin glue in vitro (x4). (A) Surviving myoblasts after three days in culture in fibrin glue. (B) Surviving myoblasts after seven days in culture in fibrin glue. (C) Dead myoblasts after three days in culture in fibrin glue. (D) Dead myoblasts after seven days in culture in fibrin glue. Note the small amount of death as a result of the surrounding matrix.
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Cell retention and survival.
After 24 h, myoblast densities in BSA and fibrin glue after injection were not significantly different (p = 0.85). Myoblasts injected in BSA comprised 15.8 ± 9.2% of the infarct, whereas myoblasts injected in fibrin glue covered 17.3 ± 14.6%. Myoblasts transplanted in fibrin glue were found both in clumps surrounded by the fibrin matrix and dispersed within its fibrils (Fig. 3). After five weeks, the myoblast density in the infarct area was significantly greater when the cells were injected in the fibrin glue than when injected in BSA (p = 0.03). Cells injected in fibrin glue covered 9.7 ± 4.2% of the infarct area compared with 4.3 ± 1.5% when injected in BSA. Transplanted myoblasts injected in BSA were most often found at the border of the infarct scar and not within the ischemic tissue at five weeks after injection (Figs. 4A and 4C). In contrast, myoblasts injected in fibrin glue were found both at the border and within the infarct scar (Figs. 4B and 4D). Cells transplanted in fibrin glue were often surrounding arterioles within the infarct scar (Figs. 4B and 4D, arrowheads). Figures 4C and 4D display the location of the normal and infarcted myocardium, thus allowing one to visualize the location of the anti-skeletal, fast MHC labeled myoblasts in Figures 4A and 4B, respectively.
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Figure 3 Myoblast in fibrin glue 24 h after injection. (A) 4',6-diamidino-2-phenylindole-labeled myoblasts (x4) injected in fibrin glue are found in the infarct wall. (B) Corresponding hematoxylin and eosin stained section (x4). Transplanted myoblasts are surrounded by fibrin glue within the infarct scar. (C) Higher-magnification hematoxylin and eosin section (x10) displaying transplanted myoblasts in fibrin glue. (D) Hematoxylin and eosin stained section (x10) of fibrin glue ex vivo.
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Figure 4 Myoblast survival and location within myocardium after five weeks (x100). (A and B) Anti-skeletal, fast myosin heavy chain-labeled myoblasts visualized with a Cy3 secondary antibody. (C and D) Hematoxylin and eosin-stained sections that neighbor (A) and (B), respectively. (C and D) Normal and infarcted myocardium are labeled to visualize the location of transplanted cells. (A) Transplanted skeletal myoblasts in bovine serum albumin are located at the border of the infarct scar. (B) In contrast, a greater number of skeletal myoblasts injected in fibrin glue are located both at the border and within the infarct scar. Surviving myoblasts are in clumps surrounding arterioles (arrows in B and D) in the scar.
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Histology.
Infarct scar size as determined by percent of the LV was measured for each group. The infarct size in the control (BSA) group was 26.5 ± 2.2%. There was no significant difference in infarct size between the three treatment groups (p = 0.45); however, both injection of fibrin glue and myoblasts in fibrin glue resulted in significantly smaller infarcts (p = 0.03 and p = 0.003, respectively) compared with a BSA control injection. Fibrin glue reduced the infarct size to 19.7 ± 3.8%, whereas myoblasts in fibrin glue reduced the size to 17.5 ± 3.4%. In contrast, myoblasts injected in BSA did not produce a statistically smaller infarct than injection of BSA (20.9 ± 5.2%; p = 0.24) (Fig. 5). A histological review of hematoxylin and eosin stained sections from each group demonstrated that there were no significant immune reactions. The scars did contain scattered hemosiderin-laden macrophages, which are evidence of previous hemorrhage, and rare mononuclear cells; however, there was virtually no active inflammation.
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Figure 5 Infarct size. The injection of fibrin glue and skeletal myoblasts resulted in statistically smaller infarcts than injection of bovine serum albumin (BSA). In contrast, the infarct size of hearts injected with skeletal myoblasts in BSA was not statistically different from hearts injected with BSA. *p < 0.05 compared with BSA (fibrin, p = 0.03; myoblasts in fibrin, p = 0.003). LV = left ventricle.
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Neovasculature formation.
To assess the angiogenic potential of fibrin glue in ischemic myocardium, infarcted rat hearts injected with fibrin glue and BSA were examined for capillary density at five weeks after injection. There was no significant difference between groups (p = 0.64). Microvessels were labeled with anti-smooth muscle actin in both the fibrin and BSA groups to determine whether fibrin glue induces formation of larger vessels. Even without treatment, collateral arterioles are often seen bordering the scar after MI; thus, separate vessel counts were performed on vessels within the infarct and those bordering the scar. Microvessel density data are summarized in Table 1. Alpha-smooth muscle actin labeled microvessel density for the total infarct area in the fibrin group was significantly greater than that in the BSA group (p = 0.004). There was no difference in vessel density bordering the infarct between the two groups (p = 0.32); however, there was a significant difference within the scar between the fibrin and BSA groups (p = 0.001). The alpha-smooth muscle actin labeled microvessel density of the two groups, including myoblasts, was also calculated. Injection of myoblasts in fibrin glue significantly increased the total and within-scar microvessel density compared with injection of myoblasts in BSA (p = 0.007 and p = 0.02, respectively). There was again no difference in alpha-smooth muscle actin labeled microvessels bordering the infarct scar (p = 0.21). We also compared the BSA group with the myoblasts in BSA group and the fibrin group with the myoblasts in fibrin group to determine whether the addition of myoblasts affected microvessel formation. The addition of myoblasts in both BSA and fibrin resulted in a significant or near-significant decrease in the total vessel density (p = 0.04 and p = 0.05, respectively). The addition of myoblasts also decreased the within-scar vessel density (p = 0.02 and p = 0.01, respectively). After the injection of fibrin glue, a large number of alpha-smooth muscle actin labeled microvessels, predominately arterioles, were found within the infarct scar (Figs. 6A and 6B). Figure 6B has been stained with hematoxylin and eosin and is the neighboring slide to Figure 6A. Normal, healthy myocardium, which is denoted by its darker staining, and the infarct scar, which is denoted by lighter staining, can both be visualized in Figure 6B. Figure 6B demonstrates that the large number of labeled arterioles in Figure 6A are in fact within the infarct scar.
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Figure 6 Fibrin-induced arteriole formation (x100). (A) Anti-smooth muscle actin-labeled arterioles visualized with a Cy3 secondary antibody. (B) Hematoxylin and eosin stained section that neighbors (A). (B) Normal, healthy myocardium is denoted by the darker staining whereas the infarct scar has lighter staining. (A and B) Fibrin induces the formation of several arterioles within the infarct.
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Discussion
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Our results indicate that cell transplant survival, but not cell retention, in infarcted myocardium is enhanced by injection of cells in fibrin glue. Injection of cells in fibrin glue did not affect the amount of myoblasts in the infarct after 24 h. These results indicate that fibrin glue does not increase cell retention. Because fibrin glue remains liquid for a few seconds, cells may continue to be squeezed out of the beating myocardium upon injection. In contrast, the area of the infarct wall covered by transplanted myoblasts after five weeks was significantly greater when the myoblasts were injected in fibrin glue, indicating that fibrin increases cell survival. Fibrin may increase cell survival by acting as a temporary extracellular matrix for the transplanted cells. Typically, cells are injected in completely liquid formulations of saline, cell culture medium, or BSA; however, fibrin glue solidifies inside the myocardium, giving the cells a temporary semi-rigid scaffold. Fibrin glue also contains arginine-glycine-asparagine (RGD) motifs and binds to cell receptors (predominately integrins) (30). Upon injection in fibrin glue, the cells are entrapped within this temporary extracellular matrix. A previous study has demonstrated that the fibrin matrix is maintained for 7 to 10 days before degradation (31), thus allowing time for the transplanted cells to form their own matrix. Fibrin glue is thus an injectable scaffold that promotes viability of cells delivered directly into the infarct wall. Another possibility for the increase in cell survival is that the injection of fibrin glue into ischemic myocardium induced neovasculature formation. An increase in blood supply would provide a less ischemic region for the cells to thrive. It has been previously reported that the injection of cells into vascularized myocardium increases survival compared with injection in ischemic myocardium (11). This is further supported by the observation that myoblasts injected in fibrin glue were often found surrounding arterioles within the infarct scar. By increasing cell transplant survival in ischemic myocardium, fibrin glue may be the necessary modification to the standard transplantation procedure. There is also the possibility that the fibrin matrix induces myoblast proliferation, which would increase the number of surviving cells in the myocardium.
We have also demonstrated that injection of fibrin glue alone as well as myoblasts in fibrin glue decreases infarct scar size. A decrease in the area covered by the scar indicates a reduction of late infarct expansion. As an indicator of negative LV remodeling, a decrease in late infarct expansion indicates that fibrin is capable of preventing negative LV remodeling after MI in rats. These histological results corroborate initial echocardiographic findings, which demonstrated that fibrin glue and cells in fibrin glue preserved wall thickness and fractional shortening (19). Fibrin may serve as an internal wall support by increasing stiffness. It may also simply affect remodeling by increasing wall thickness. Although there was no significant difference in infarct size among the three treatment groups, injection of skeletal myoblasts in BSA did not produce a statistically smaller infarct than the control injection, consistent with previous reports of transplantation survival problems within infarcted myocardium (5,8,11,12). This trend indicates that fibrin and myoblasts in fibrin glue may have the potential to produce smaller infarcts than injection of myoblasts in BSA. The injection of myoblasts in BSA may not be capable of producing a large enough graft to reduce infarct size.
Fibrin glue also induced alpha-smooth muscle actin labeled microvessel formation within the infarct scar, thus suggesting an increase in blood supply to the ischemic myocardium. Although larger alpha-smooth muscle actin labeled microvessels may be venules, the majority of vessels would be arterioles. It is significant that fibrin glue results in arteriogenesis since formation of solely capillaries does not necessarily indicate an increase in blood flow, due to the ease of regression of vessels without smooth muscle (32). Fibrin did not, however, increase capillary formation compared with the injection of BSA. Injection into the myocardium is known to induce some angiogenesis. Fibrin may therefore not be capable of producing a greater angiogenic response. In addition, the increase in larger vessels may be a result of the differentiation of existent capillaries into larger-diameter arterioles. Natively, fibrin is highly involved in wound healing and acts as the body's natural matrix for angiogenesis. Endothelial cells migrate through the fibrin clot via  vß3 integrin binding to arginine-glycine-asparagine motifs in fibrin (33). The production of plasmin at the location of migrating endothelial cells degrades the fibrin matrix. This decrease in fibrin density allows for capillary tube formation (34). As the cells migrate through the less dense fibrin, they interact with residues on the beta chain of fibrin via vascular endothelial cadherins and promote capillary morphogenesis (35). In addition to providing a matrix for endothelial cell migration and capillary tube formation, fibrin also acts as a sustained-release reservoir for several growth factors (36) and fibrinolytic enzymes (37). A degradation product of fibrin, fibrin fragment E, also has been shown to induce angiogenesis in the chick chorioallantoic membrane assay (15), and stimulate proliferation, migration, and differentiation of human microvascular endothelial cells (16), and stimulate migration and proliferation of smooth muscle cells (17). The administration of exogenous fibrin into the subcutaneous space of guinea pigs also has been shown to induce angiogenesis (18).
Fibrin glue is an already Food and Drug Administration-approved biomaterial that is routinely used as a surgical adhesive and sealant. This biopolymer is formed by the addition of thrombin to fibrinogen. Thrombin enzymatically cleaves fibrinogen, which then forms the polymer fibrin. After combination of the two components, the solution remains liquid for several seconds before polymerizing. Fibrin glue could therefore be delivered to the myocardium via catheter in humans, thus requiring only a minimally invasive procedure; however, certain precautions must be taken to avoid injection into the LV cavity or coronary vessels, which could result in an embolic event. It is also biocompatible and non-toxic and does not induce inflammation, foreign body reactions, tissue necrosis, or extensive fibrosis (38). Our laboratory has also demonstrated that the injection of fibrin glue alone or skeletal myoblasts in fibrin glue prevents the deterioration of cardiac function and infarct wall thinning after MI in rats (19). Although this initial work has been performed with skeletal myoblasts, we anticipate that fibrin glue would improve cell viability of other transplanted cells such as stem cells.
Study limitations.
One limitation of the animals used in this study is that they were not an inbred strain and an allograft transplantation was performed; thus, graft rejection is expected to be higher. Our preliminary results with fibrin glue and myoblasts indicated that viable grafts survive in Sprague-Dawley rats. Sprague-Dawley rats represent a "worst-case" scenario for cell survival because of their increased immune reaction. If fibrin glue is capable of increasing graft size in this "worst-case" scenario, we anticipate that we would find a more dramatic effect in an inbred strain.
Conclusions.
This study indicates that fibrin glue may have potential in the treatment of patients suffering from MI. It may be a possible treatment that increases neovasculature formation and decreases infarct size or a possible novel method for increasing cell transplant graft size in ischemic myocardium.
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Footnotes
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This research was supported by a grant from the Nora Eccles Treadwell Foundation.
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