Posts Tagged ‘Infarction’

Neoangiogenic Effect of Grafting an Acellular 3-Dimensional Collagen Scaffold Onto Myocardium

Author: Larry H. Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN


This is Part 3 of a series of contributions on cardiac regeneration after myocardial infarct with stem cells.

Progenitor Cell Transplant for MI and Cardiogenesis (Part 1)
Larry H. Bernstein, MD, FCAP, and Aviva Lev-Ari, PhD, RN,-and-cardiogenesis/

Source of Stem Cells to Ameliorate Damage Myocardium (Part 2)
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN Damaged_Myocardium/

An Acellular 3-Dimensional Collagen Scaffold  Induces Neo-angiogenesis (Part 3)
Larry H. Bernstein, MD, FCAP, and Aviva Lev-Ari, PhD, RN _Induces_Neo-angiogenesis/

This series of articles discusses the difficulties we have encountered in adopting stem cell research to clinical therapeutics in regeneration of cardiac tissue damaged post myocardial infarct.  Enormous problems have been encountered in the selection of progenitor cells, the growth into compatible and functional myocardial tissue, and the survival of the myocardium.  Part I went into some detail on a method of obtaining suitable cells, growing them in sheets, and transferring the sheets to the surface for regeneration and repair, which is now going into clinical trials.  Part I will be confined to the importance of source of progenitor cells, whether adult stem cells or umbilical cord blood.

These are issues that need to be considered

  • Adult stem cells
  • Umbilical cord tissue sourced cells
  • Sheets of stem cells
  • Available arterial supply at the margins
  • Infarct diameter
  • Depth of ischemic necrosis
  • Distribution of stroke pressure
  • Stroke volume
  • Mean Arterial Pressure (MAP)
  • Location of infarct
  • Ratio of myocytes to fibrocytes
  • Coexisting heart disease and, or
  • Comorbidities predisposing to cardiovascular disease, hypertension
  • Inflammatory reaction against the graft

Despite successes in pre-clinical animal models with stem cells, a problem arises with respect to the biology of the transplanted progenitor cells.  In Part II, we discovered that neo-angiogenesis occurs without evidence of myocyte generation.  That is the topic we discuss here.

Grafting an Acellular 3-Dimensional Collagen Scaffold Onto a Non-transmural Infarcted Myocardium Induces Neo-angiogenesis and Reduces Cardiac Remodeling

MA Gaballa,a JNE Sunkomat,a H Thai,a,b E Morkin,a G Ewy,a and S Goldman,a,b
From the aSection of Cardiology, University of Arizona Sarver Heart Center, Tucson, Arizona and bSouthern Arizona Veterans Administration Health Care System, Tucson, Arizona.
J Heart Lung Transplant 2006; 25: 946–54.

Background: This study was designed to determine whether tissue engineering could be used to reduce ventricular remodeling in a rat model of non-transmural, non–ST-elevation myocardial infarction.

Methods: We grafted an acellular 3-dimensional (3D) collagen type 1 scaffold (solid porous foam) onto infarcted myocardium in rats. Three weeks after grafting, the scaffold was integrated into the myocardium and retarded cardiac remodeling by reducing left ventricular (LV) dilation. The LV inner and outer diameters, measured at the equator at zero LV pressure, decreased (p < 0.05) from 11,040 ± 212 to 9,144 ± 135 pm, and 13,469 ± 187 to 11,673 ± 104 pm (N = 12), after scaffold transplantation onto infarcted myocardium. The scaffold also shifted the LV pressure–volume curve to the left toward control and induced neo-angiogenesis (700 ± 25 vs 75 ± 11 neo-vessels/cm2, N = 5, p < 0.05). These vessels (75 ± 11%) ranged in diameter from 25 to 100 pm and connected to the native coronary vasculature. Systemic treatment with granulocyte-colony stimulating factor (G-CSF), 50 pg/kg/day for 5 days immediately after myocardial injury, increased (p < 0.05) neo-vascular density from 700 ± 25 to 978 ± 57 neo-vessels/cm2.

Conclusions: A 3D collagen type 1 scaffold grafted onto an injured myocardium induced neo-vessel formation and reduced LV remodeling. Treatment with G-CSF further increased the number of vessels in the myocardium, possibly due to mobilization of bone marrow cells.


Despite advances in the treatment of heart failure after myocardial infarction, the incidence and prevalence of this disease is increasing steadily. This is due in part to recent advances in treatment of the acute ischemic event; however, even when patients survive a large myocardial infarction, they are left with damaged ventricles, often leading to heart failure without another ischemic event. Cardiomyocytes may not possess sufficient regenerative capacity after birth because loss of these cells in the acute setting results in a fibrous scar and associated regional contractile dysfunction. Transplantation of exogenous cardiac or stem/progenitor cells has been proposed as treatment for heart failure.1,2 Despite the apparent success of stem-cell therapy, there are conflicting reports about the fate of these cells and their effects on cardiac function.3–6 Previous studies in tissue engineering show that grafting an alginate or collagen scaffold seeded with either fetal cardiomyocytes or fibroblasts on injured myocardium induces neovascularization, but the morphology of these new vessels is abnormal.7–9 Our hypothesis is that grafting of a biodegradable 3D collagen type 1 scaffold onto infarcted rat myocardium would provide temporary mechanical support for the ventricular wall, induce neovascularization, and reduce cardiac remodeling.

We used the cryoinjury approach to create a model of a non–ST-elevation myocardial infarction (NON-STEMI). Our model of a relatively small non-transmural injury is similar to what is seen clinically in patients with acute ischemic injury. We speculate that the scaffold provides an initial mechanical support to retard myocardial dila¬tion after acute MI and a later collateralization between native viable blood vessels in the injured myocardium and the newly formed vessels within the scaffold. Because mobilization of progenitor/stem cells using granulocyte-colony stimulating factor (G-CSF) has been reported to increase vascularization and improve car diac function after MI,10 G-CSF treatment is performed at the time of grafting to further enhance neo-vascular-ization in our model. This study was designed as a “proof of concept” with the ultimate clinical goal to surgically graft a matrix onto the heart in patients with acute MI. This could be done using a minimally invasive approach, such as video-assisted thoracic surgery (VATS) with or without robotics.


Six-month-old adult male Fisher 344 rats (inbred strain) were used in this study.6 Fifty infarcted and 10 non-injured rats were used. Six groups of rats were studied:
(1) sham (n = 5);
(2) cryoinjury (n = 14);
(3) sham scaffold (n = 5);
(4) infarction scaffold (n = 12);
(5) infarction G-CSF (n = 12); and
(6) infarction scaffold G-CSF (n = 12).

Scaffolds were grafted immediately after cryoinjury. Six weeks after grafting –

  • hemodynamics,
  • LV pressure–volume,
  • vascular density,
  • immunohistochemistry and
  • LV remodeling measurements were performed.

The experimental protocol was carried out as described in what follows.

Experimental Myocardial Infarction Model

Myocardial infarction was produced as previously de-scribed.11 In brief, after anesthesia with ketamine, xylazine, acepromazine and atropine, a left lateral thoracotomy was performed through the third and fourth intercostal space. A 4-mm stainless-steel probe was submersed in liquid nitrogen and placed on the LV free-wall for 10 seconds. The heart was assessed visibly for viability, and the injury procedure was repeated once at the same site. Before closing the chest, the collagen scaffold was engrafted onto the injured myocardium as described in what follows. The lungs were inflated, the chest was closed, and the animal was allowed to recover. In the sham rats the chest was opened but the cold probe was not placed on the myocardium.

Grafting of 3D Collagen Type 1 Scaffold

Immediately after infarction, while the chest was still open, the scaffold was sutured to the injured myocardium at four different points along the outer boundary of the scaffold. The scaffold, a circular collagen type 1 foam disk 5 mm in diameter and 0.5 mm in thickness (Suwelack Co., Billerback, Germany), was prepared from porcine skin collagen. The scaffold is highly flexible with a porosity of 70% and an average pore diameter of 30 to 60 m as determined by scanning electron microscopy. Di-isocyanate was used to elimi-nate toxic residue after cross-linking, which is common with the use of glutaraldhyde polymers. Before engrafting, the scaffold was washed with distilled water, allowed to dry overnight, treated with rat serum, and again allowed to dry overnight.

Cytokine Treatment

Immediately after scaffold grafting, rats in Groups 5 and 6 received subcutaneous injection of G-CSF (50 g/kg/ day; Amgen Biologicals) for 5 days. Control rats and Groups 2 and 4 were treated with saline. Rats were studied 6 weeks after grafting.

All of the following measurements were performed at 6 weeks after grafting and in all groups, unless otherwise specified.


Six weeks after grafting, rats were anesthetized with inactin (100 mg/kg intraperitoneal injection) and placed on a specially equipped operating table with a heating pad to maintain constant body temperature. After endotracheal intubation and placement on a rodent ventilator (Harvard Instruments), a 2F solid-state micromanometer-tipped catheter with two pressure sensors (Millar) was inserted via the right femoral artery, with one sensor located in the left ventricle and another in the ascending aorta. The pressure sensor was equilibrated in 37°C saline before obtaining baseline pressure measurements. After a period of stabilization, LV and aortic pressures and heart rate were recorded and digitized at a rate of 1,000 Hz using a PC equipped with an analog-to-digital converter and customized soft-ware. From these data, LV dP/dt was calculated.

Left Ventricular Pressure–Volume Relationships

Six weeks after grafting and after completing the hemodynamic measurements, LV pressure–volume relations were measured in four randomly selected rats from each group as outlined in our previous publications.12 In brief, a catheter, consisting of PE-90 tubing with telescoped PE-10 tubing inside and a water-filled bal-loon attached, was inserted in the left ventricle via the left atrial appendage. Previous testing of the balloon showed essentially infinite compliance up to 0.68 ml of volume; thus, all LV infusions were kept at 0.68 ml. One end of the double-lumen LV catheter was con-nected to a volume infusion pump (Harvard Apparatus) while the other end was connected to a pressure transducer zeroed at the level of the heart. A drainage tube was also placed in the LV cavity, and the right ventricle was partially incised to prevent loading on the LV. After 2 minutes of perfusion with phosphate-buffered saline (PBS), the LV was filled (0.6 ml/min) and unfilled while pressure was recorded onto a physiologic recorder (Gould). Volume infused is a function of filling rate. The ventricle was infused to 60 mm Hg for all experiments and recordings were done in triplicate.

Communication Between the Newly Formed Vessels Within the Scaffold and Native Vessels in Surviving Myocardium

To determine whether the newly formed vessels within the scaffold were connected to the native circulation in the surviving myocardium, isolated hearts were perfused with Evans blue at the aortic root. In brief, the hearts were perfused at 100 mm Hg with PBS just to clear the blood from the coronary circulation. The hearts were then perfused at 100 mm Hg with 4 mg/ml Evans blue in PBS for 30 seconds. Standard 35-mm photographs were taken within the first 1 to 1.5 minutes after starting Evans blue perfusion. The hearts were than washed with PBS and used for morphologic and histologic analysis as described in what follows.

Morphology, Histology and Immunohistochemistry

LV remodeling (morphology).
After completing all the aforementioned measurements, the hearts were per-fused-fixed with glutaraldhyde at 100 mm Hg via the coronary circulation at zero LV pressure. In the in-farcted group, the lesion area, which is slightly larger than the steel-probe cross-sectional area, was visually measured using standard techniques developed in our laboratory to measure infarct size.13,14 However, in the infarction scaffold groups, it was difficult to measure the lesion area 6 weeks after grafting because the scaffolds were absorbed by the heart tissue and it was difficult to distinguish between the scaffold tissue and the scarred area. Each heart was cut in the short axis to five segments from the apex to the base. The inner and outer diameters were measured in the segment located at the short-axis equator using a computer attached to a digital camera.14
Vascular density within the scaffold.
The perfused-fixed hearts were dehydrated and embedded in paraffin. Five-micron-thick transverse sections, which included the scaffold, were processed for hematoxylin–eosin staining. Selected sections were stained using Factor VIII–like antigen (von Willebrand factor) to identify the endothelial cells. Sections were re-hydrated and antigen retrieval was accomplished by incubation twice in 10 mmol/liter citric acid (pH 6.0) at 95°C for 5 minutes. Endogenous peroxidase activities were removed by incubation for 10 minutes in a PBS solution containing 0.6% H2O2. Slides were incubated with primary antibody and biotinylated rabbit anti-rat IgG (Dako) as secondary antibody. After rinsing with PBS, 0.05% diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide were applied for 5 minutes and washed with water. Muscle sections were examined for positive (brown color) staining. Vascular density was measured by light microscopy at x40 magnification. The number of cross-section vessels per field was counted. Average measurements from six different fields were recorded for each value. Knowing the area of the optical field, data were reported as number of vessels/mm2.
Vascular smooth muscle cells.
Vascular smooth muscle cells were detected by immunohistochemical (IHC) analysis in selected myocardial sections, using antibody directed against -smooth-muscle actin (M0951, Dako).15 Tissue fixation and antibody incubation and detection were performed as described earlier for the vascular density measurements.

Cardiac myocytes.

To determine whether the cells that migrated into the scaffold exhibit a cardiomyocyte-like phenotype, myocardial sections including the scaffold were incubated with mouse monoclonal IgGs primary antibody against either sarcomeric myosin heavy chain, MF20 (1:100 dilution, hybridoma supernatant; Hybrid-oma Bank, University of Iowa) or cardiac troponin T-C (Santa Cruz Biotechnology). Immunostaining on deparaffinized sections was performed using peroxidase standard protocols (as described earlier).

Statistical Analysis

Data are presented as mean +/- SD.  p  < 0.05 indicates statistical significance. For Groups 1, 2, 3 and 4, a 2-way analysis of variance (ANOVA; injury and scaffold-graft¬ing as the 2 factors) was performed, followed by multiple comparisons using Student–Newman–Keuls test. A second 2-way ANOVA (scaffold grafting and G-CSF treatment as the 2 factors), followed by multiple comparisons using Student–Newman–Keuls test, was performed on Groups 2, 4, 5 and 6.


A total of 50 infarcted and 10 non-infarcted rats were used in this study. Immediately after injury, the 3D collagen type 1 scaffold was grafted onto the infarcted myocardium. All major findings of this study were obtained at 6 weeks after scaffold grafting. In a small sub-set of animals, the scaffold was examined at 3 weeks after grafting and was found to be integrated (i.e., attached to the underlying myocardium), not only at the four suture points along the perimeter of the scaffold, but also in the middle section of the scaffold (n =2). Six weeks after grafting, when all subsequent measurements were obtained, the scaffold was mostly absorbed by the underlying myocardial tissue and the distinction between the scaffold tissue and the underlying scar became difficult to identify (n = 36).

Hemodynamics and LV Remodeling After Scaffold Grafting

Non-transmural injury resulted in LV dilation. Six weeks after infarction the LV lumen and the outer diameters, measured at the equator at zero LV pressure, were increased (p < 0.05) from 8,726 ± 189 to 11,041 ± 212 um, and 12,006 ± 99 to 13,469 ± 189 um, respectively (N = 12). Six weeks after scaffold transplantation onto infarcted myocardium, reduced myocardial dilation was detected. The LV inner lumen diameter (Di) and the outer diameter (Do) measured at the short-axis equator at zero LV pressure were decreased from 11,041 ± 212 to 9,144 ± 135 um (N = 12, p < 0.05) and from 13,469 ± 187 to 11,673 ± 104 um (N = 12, p < 0.05), respectively. The scaffold also improved cardiac remodeling by shifting of the LV pressure–volume curve to the left toward the sham (control) curve (Figure 1). In this study, the extent of damage by cryoinjury was small, with no changes in hemodynamic parameters in the injured rats with or without the scaffold at 6 weeks after grafting. Specifically, there were no changes  (nopt shown)

  • in LV end-diastolic pressure,
  • mean arterial pressure or
  • LV dP/dt (Table 1).

Figure 1. Pressure–volume curves for the four groups

Figure 1. Pressure–volume curves for the four groups. Solid line: treatment group (infarcted rats with collagen scaffold); dashed line: untreated sham and sham treated with collagen scaffold; dotted line: untreated infarcted groups (cryoinjury). The curve for sham with collagen scaffold is superimposed upon the untreated sham curve. Note that, 6 weeks after transplantation, the P-V curve is shifted to the left. N = 4 for each group. *p < 0.05.

Induction of Large Vessel Formation 6 Weeks After 3D Collagen Type 1 Scaffold Transplantation

Six weeks after grafting the collagen scaffold onto the infarcted myocardium, large vessels within the graft were observed (Figure 2A). These vessels differed from the typical angiogenesis achieved during wound heal¬ing, which is characterized by thin-walled, leaky vessels (Figure 2B). Vascular density was measured by counting the number of Factor VIII positively stained cells (Figure 3). This microscopic evaluation was carried out by a histologist without knowledge of the intervention. The scaffold induced neo-angiogenesis (700 ± 25 vs 75 ± 11 neo-vessels/cm2, N = 5). These vessels (75 ± 11%) ranged in diameter from 25 to 100 um. Note the presence of mural cells within the vessel wall, which were positive for a-smooth-muscle actin (Figure 4). Finally, the scaffold transplantation onto infarcted hearts decreased (p < 0.05) the scar area (12 ± 3% vs 21 ± 8%, N = 8) compared with infarction alone. However, it was difficult to distinguish the scar tissue from the scaffold at 6 weeks after grafting.

Figure 2. Engrafted scaffold showing vessels

Figure 2. (A) High magnification (original magnification 40) of the H&E stain of the engrafted scaffold showing large vessel (arrows). These vessels are thick-walled and have multiple cell layers. (B) Same magnification as (A) of the H&E staining of infarcted myocardium without the scaffold. Note that the ischemia-induced vascularization is characterized by thin-walled vessels.

Figure 3.  Neovascularization in scaffold

Figure 3. (A) A typical Factor VII staining for endothelial cells in cryoinjured heart with scaffold shows neo-vascularization in the scaffold (top) at 6 weeks after grating onto native myocardium (bottom). (B) A different field from the same section.

Figure 4. Smooth muscle actin staining

Figure 4. Vascular a-smooth-muscle actin staining. (A) Native (non-injured) myocardium (control). (B) Scaffold. Brown staining within the neo-vessels indicates the presence of mural cells 6 weeks after grafting.

Effects of Cytokine Treatment on Vessel Formation 6 Weeks After Scaffold Engraftment

Comparing infarcted + scaffold to infarcted + scaffold + G-CSF rats, systemic treatment with G-CSF 50 ug/kg/ day for 5 days started immediately after cryoinjury increased (p < 0.05) neo-vascular density within the scaffold from 700 ± 25 to 978 ± 57 neo-vessels/mm2 (Figure 5). No effects were observed for systemic treatment with G-CSF in LV remodeling or pressure– volume (P-V) curves when infarcted + scaffold + G-CSF were compared with untreated infarcted + scaffold rats.

Figure 5. Effects of scaffold grafting with and without G-CSF

Figure 5. Effects of scaffold grafting, with and without G-CSF administration, on vascular density. Vascular density increased by 8-fold with the scaffold alone and by 40% with scaffold and G-CSF treatment. *p < 0.05 infarcted scaffold compared with infarcted alone. **p < 0.05 infarcted scaffold G-CSF compared with infarcted scaffold. N  = 5 for each group.

Communication Between Newly Formed Vessels Within the Scaffold and Native Coronary Circulation

In both infarcted + scaffold and cryoinjured + scaffold + G-CSF groups, 6 weeks after grafting, isolated hearts, perfused with Evans blue at the aortic root, showed that the newly formed vessels within the scaffold were connected to the native vessels in the surviving myo-cardium, as indicated by the presence of blue dye within the scaffold (Figure 6B). To confirm if the newly formed vessels within the scaffold are connected to the native coronary circulation, hearts perfused with Evans blue were sectioned (5 um), hematoxylin–eosin (H&E)-stained, and examined under a fluorescence micros-copy. Evans blue showed red under fluorescence (white arrows, Figure 7).

Figure 6. coronary artery prfusion of isolated hearts

Figure 6. Coronary artery perfusion of isolated hearts with Evans blue. (A) Infarcted heart without scaffold (control). (B) Infarcted heart with scaffold. Note the neovasculature within the scaffold that perfuses blue, indicating a connection to the coronary arteries.

not shown

Figure 7. Micrographs showing Evans blue within myocardial vessels (white arrows, red color). H&E myocardial sections examined under fluorescence microscopy (original magnification 40). (A) Non-infarcted myocardium. (B) 3D scaffold.

Induction of Myofibril-like Tissue Within the Scaffold 6 Weeks After Scaffold Engraftment

Six weeks after collagen scaffold grafting onto infarcted myocardium and after treatment with G-CSF for 5 days, there was some evidence of a limited number of myofibril-like cells identified within the scaffold (Figure 8A). These myofibril-like cells were positive for the sarcomeric myosin heavy chain antibody (Figure 8A), MF20 (Hybridoma Bank, University of Iowa) and car-diac troponin T-C (sc-8121, Santa Cruz Biotechnology; Figure 8B). In that section, where these myofibril-like cells are found, there was < 0.01% per field.

Figure 8. cardiac myofibril bundle in scaffold

Figure 8. (A) Detection of cardiac myofibril bundle within the scaffold (left) by MF20 (A) and cardiac troponin T (B) immunohistochemical staining (brown, arrows) in the infarcted scaffold groups. Native myocardium is shown on the right. (C) Control staining for sham rats (uninjured myocardium).


In the present study we have shown that, in the presence of a non-transmural MI:

(1) grafting of a 3D extracellular matrix scaffold onto injured myocardium results in neo-vascularization and reduces cardiac re-modeling;

(2) mobilization of bone marrow cells using cytokine treatment further increases this neo-vascularization; and

(3) the resulting vasculature consists of large vessels, which are connected to the native coronary circulation in the surviving myocardium.

The further increase in neo-vascularization by G-CSF treatment suggests that bone marrow cells may contribute to this process. This report shows that, as a “proof of principle,” it is possible to graft a biodegradable scaffold matrix onto an injured heart to promote neo-vascularization and to possibly provide a stable platform in which circulating and/or resident progenitor cells can flourish.
We used an infarcted non-transmural MI model to examine neovascularization at the early stages of an ischemic injury that occurs without severe hemodynamic insult. The clinical correlate of our model is the non–ST-elevation MI (NON-STEMI). Interestingly, similar to our experimental model, in this clinical infarction model

  • there is LV remodeling with chamber dilation without changes in hemodynamics.

The finding that the collagen scaffold prevents this remodeling suggests that this type of approach may have a role in the treatment of early stages of ischemic injury.
Our extracellular matrix scaffold

  • induced large vessels containing vascular smooth muscle cells as evidenced by α-smooth-muscle actin (α-SMA)-positive staining.

These data differ from a previous report, which showed that grafting a scaffold based on a 3D human fibroblast patch on infarcted myocardium induced thin-walled vessels.8 The difference between these two approaches may be due in part to the scaffold itself. The scaffold we used is highly flexible with a moderate pore size (30 to 60 m) and high porosity (70%), thus allowing for cell attachment, migration, delivery of nutrients and waste removal. It also has the advantage that cells and/or growth factors can be delivered in a controlled setting before grafting. More importantly, cyclic stretch applied to the scaffold in vivo, during the cardiac cycle, may help explain the induction of large vessels within the scaffold.
In our NON-STEMI model, adverse remodeling occurs without major hemodynamic insult. The grafted scaffold

  • prevents LV dilation and thinning of the infarcted myocardium.

Preservation of LV geometry may be the main mechanism of the improved P-V relationship after grafting. The scaffold may act as a temporary mechanical support for the injured ventricular wall. Theoretically, the scaffold could also act as a homing site for the injury-mobilized cells that may reduce cardiac remodeling by induction of neovascularization. This is consistent with a previous report in which neovascularization has been suggested to improve cardiac remodeling and function.16
Several different types of myocyte preparations have been directly injected into the myocardium, such as

  • smooth muscle cells,
  • skeletal muscle cells and
  • satellite skeletal muscle cells,

all of which have been shown to enhance cardiac function.

  1. autologous transplantation of skeletal muscle has been shown to reverse LV remodeling.17–19
    1. this approach has been complicated by the induction of arrhythmias that may be due to the lack of electromechanical coupling between the injected skeletal muscle cells and the native myocardium.20
    2. repopulate the infarcted myocardium by direct injection with the patient’s own bone marrow progenitor cells.2,21–23

While these reports have even led to preliminary clinical trials,23 the fate of exogenously delivered cells directly into the myocardium is still unclear.5,6 It is beyond the scope of this report to reconcile this debate. Recently, intramyocardial transplantation of a pouch containing a mixture of collagen type 1 gel and embryonic stem cells was reported to restore infarcted myocardium.24 The approach outlined here for the heart is analogous to the reports of bone marrow cells contributing to the endothelialization of vascular autografts.25
In the present study, we grafted a 3D collagen type 1 scaffold onto the myocardium immediately after cryoinjury. This model was purposely chosen over infarction by coronary artery ligation because cryoinjury with our technique creates a well-defined, non-transmural, reproducible, similarly sized scar every time, as opposed to the coronary ligation model, in which the infarct size is transmural and variable depending upon how proximal the ligature is on the coronary artery. Cryoinjury also results in a non-transmural necrosis, potentially

  • allowing the still-viable native circulation in the surviving myocardium underneath the scar to connect to the newly formed vessels within the scaffold.

The scaffold was applied soon after infarction because we believe that injury is a strong stimulus for recruiting cells into the scaffold in vivo. After acute MI, mRNA expressions of cytokines, such as vascular endothelial growth factor (VEGF), flk-1 and flt-1, are elevated initially throughout the entire heart.27 Our finding of increased density of neo-vessels in the infarcted myocardium with the scaffold is consistent with data showing that the number of circulating endothelial progenitor cells increases after myocardial injury.28
The fact that we could increase the level of neovascularization with G-CSF suggests that mobilization of bone marrow cells and possible migration to the injured myocardium may be responsible for the increase in neovascularization. Although this relationship has not been directly tested in the current study, it is consistent with previous studies demonstrating that exogenous administration of cytokines such as VEGF, stromal cell–derived factor (SDF-1) and fibroblast growth factor (FGF-1)

  1. increase the number of circulating endothelial progenitor cells,
  2. their recruitment to sites of active inflammation, and
  3. induction of angiogenesis.29,30

Taken together, this study has shown that a 3D collagen type 1 scaffold immediately grafted onto an acutely injured myocardium

  • integrates with the tissue;
  • allows for cell population,
  • growth and differentiation;
  • induces large-vessel formation within the graft; and
  • retards LV remodeling.

The further increase in neovascularization after cytokine treatment with G-CSF suggests that mobilized bone marrow cells contribute to this process. Several pre-clinical and clinical trials reported beneficial effects of cell-based therapy after MI. However, due to lack of standardization (i.e., different cell type, cell number, route of administration, etc.) in both clinical and pre-clinical studies, the efficacy of these trials is still unclear. We have shown that grafting of a biodegradable scaffold may be an effective approach for cardiac re-vascularization. Our scaffold could provide a supporting structure with the appropriate milieu for new blood vessel growth.


1. Sunkomat JN, Gaballa MA. Stem cell therapy in ischemic heart disease. Cardiovasc Drug Rev 2003;21:327–42.

2. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–5.

3. Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoi-etic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004;428:664–8.

4. Balsam LB, Wagers AJ, Christensen JL, et al. Haematopoi-etic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004;428:668–73.

5. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 2001;33:907–21.

6. Kajstura J, Rota M, Whang B, et al. Bone marrow cells differentiate in cardiac cell lineages after infarction inde-pendently of cell fusion. Circ Res 2005;96:127–37.

7. Leo J, Aboulafia-Etzion S, Dar A, et al. Bioengineered cardiac grafts: a new approach to repair the infarcted myocardium? Circulation 2000;102(suppl III):III-56–61.

8. Kellar RS, Landeen LK, Shepherd BR, Naughtom GK, Ratcliffe A, Willams SK. Scaffold-based 3-D human fibro¬blast culture provides a structural matrix that support angiogenesis in infarcted heart tissue. Circulation 2001; 104:2063–8.

9. Nugent HM, Edelman ER. Tissue engineering therapy for cardiovascular disease. Circ Res 2003;92:1068–78.

10. Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA 2001;98:10344–9.

11. Li RK, Jia ZQ, Weisel RD, et al. Cardiomyocyte transplan¬tation improves heart function. Ann Thorac Surg 1996; 62:654–61.

12. Raya TE, Gaballa M, Anderson P, Goldman S. Left ventric¬ular function and remodeling after myocardial infarction in aging rats. Am J Physiol 1997;273:H2652–8.

13. Gaballa MA, Raya TE, Goldman S. Large artery remodeling after myocardial infarction. Am J Physiol 1995;268: H2092–3.



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