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Three-Dimensional Fibroblast Matrix Improves Left Ventricular Function post MI

Curators: Larry H. Bernstein, MD. FCAP and Aviva Lev-Ari, PhD, RN

Implantation of a Three-Dimensional Fibroblast Matrix Improves Left Ventricular Function and Blood Flow After Acute Myocardial Infarction

Hoang M. Thai*, Elizabeth Juneman*, Jordan Lancaster*, Tracy Hagerty*, Rose Do*, Lisa Castellano*, Robert Kellar†, Stuart Williams†, Gulshan Sethi*, Monika Schmelz*, Mohamed Gaballa*,†, and Steven Goldman*
*Section of Cardiology, Department of Medicine and Pathology, Southern Arizona VA Health Care System, Sarver Heart Center, University of Arizona, Tucson, AZ,  †Theregen Inc., San Francisco, CA

Cell Transplant. 2009 ; 18(3): 283–295.  http://dx.doi.org/10.3727/096368909788535004

Abstract

This study was designed to determine if a viable biodegradable three-dimensional fibroblast construct (3DFC) patch implanted on the left ventricle after myocardial infarction (MI) improves left ventricular (LV) function and blood flow. We ligated the left coronary artery of adult male Sprague-Dawley rats and implanted the 3DFC at the time of the infarct. Three weeks after MI, the 3DFC improved LV systolic function by increasing (p < 0.05) ejection fraction (37 ± 3% to 62 ± 5%), increasing regional systolic displacement of the infarcted wall (0.04 ± 0.02 to 0.11 ± 0.03 cm), and shifting the passive LV diastolic pressure volume relationship toward the pressure axis. The 3FDC improved LV remodeling by decreasing (p < 0.05) LV end-systolic and end-diastolic diameters with no change in LV systolic pressure. The 3DFC did not change LV end-diastolic pressure (LV EDP; 25 ± 2 vs. 23 ± 2 mmHg) but the addition of captopril (2mg/L drinking water) lowered (p < 0.05) LV EDP to 12.9 ± 2.5 mmHg and shifted the pressure–volume relationship toward the pressure axis and decreased (p < 0.05) the LV operating end-diastolic volume from 0.49 ± 0.02 to 0.34 ± 0.03 ml. The 3DFC increased myocardial blood flow to the infarcted anterior wall after MI over threefold (p < 0.05). This biodegradable 3DFC patch improves LV function and myocardial blood flow 3 weeks after MI. This is a potentially new approach to cell-based therapy for heart failure after MI.

Three-Dimensional Fibroblast Patch

Our hypothesis is that the lack of survival of new cells directly injected into the heart is related, in part, to an inadequate blood supply and inadequate matrix support for the new cells. The injected cells are fragile, resulting in cell aggregation due to lack of physical support for the cells to attach to the tissue extracellular matrix. This three-dimensional scaffold offers a potential solution to the problem of an inadequate support structure. While injection of passive materials has been proposed to improve EF potentially by decreasing wall stress (11,35), the 3DFC provides a viable cell matrix that supports new blood vessel growth (15,16). This viable cellular matrix is important because in addition to providing a new support structure for the damaged heart, we also need to create a mature blood supply such that new viable cardiac muscle can be organized in parallel forming physical and neural connections that will conduct electrical signals and create synchronized contractions. Investigators have proposed that the ideal scaffold structure for the heart would consist mainly of highly interconnected pores with a diameter of at least 200 µm, the average size of a capillary, to permit blood vessel penetration and cell interactions (5).

The 3DFC is a viable construct composed of a matrix embedded with human newborn dermal fibroblasts cultured in vitro onto a bioabsorbable mesh to produce living, metabolically active tissue (15,16) (see Fig. 1 and Fig 2). As the fibroblasts proliferate across the mesh, they secrete human dermal collagen, fibronectin, and glycosaminoglycans (GAGs), embedding themselves in a self-produced dermal matrix. The fibroblast cells produce angiogenic growth factors: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and angiopoietin-1. The construct is grown in medium supplemented with serum and ascorbate; at harvest, the medium is replaced with a 10% DMSO-based cryoprotectant, the tissue is frozen and stored at −70°C. This cryopreservation and rewarming technique has been extensively studied to ensure viability of the patch. Although the mechanisms of action of the 3DFC are not completely understood, new blood vessel growth has been documented previously in SCID mice (15).

Previous work using the 3DFC as a patch for the infarcted heart in SCID mice showed histological evidence of new blood vessel growth and improvements in global LV function using a conductance catheter (16). Our data show increases in myocardial blood flow in the infarcted heart, confirming that these blood vessels are functional and that they connect to the native myocardium. We used echocardiography to document improvements in global and regional LV function. The improvements in regional LV function are important because recent work suggests that the injection of passive materials alone may be enough to reduce wall stress and increase global EF (35). In order to prove that cell-based therapy is affecting more than a passive response, the point has been made that it is necessary to be able to define regional changes in the area of the infarcted myocardium (11). We have done this using echocardiography to document that the 3DFC increases systolic displacement of the infarcted regional anterior wall (Fig. 5). Although the mechanism of action of the 3DFC has not been completely delineated, the viable fibroblasts secrete a number of growth factors, thus providing a paracrine effect to stimulate new blood vessel growth. The vicryl mesh is biodegradable such that, with dissolution, the new blood vessel growth is in the previously damaged myocardium. The most likely explanation for the improvements in regional systolic displacement of the anterior wall is that the increases in myocardial blood flow in the border zone results in recruitment of hibernating or stunned cardiac myocytes.

The fact that the 3DFC is viable with fibroblasts implanted on a mesh is important. There are data showing that inert biodegradable patches are beneficial in treating heart failure. In our laboratory we have shown that an inert biodegradable collagen patch placed on the rat heart after a nontransmural MI improves LV function and prevents adverse LV remodeling (10). There are clinical trials with a collagen type 1 matrix seeded with autologous bone marrow cells in patients undergoing coronary artery bypass surgery (4). The best known implanted mechanical constraint device is the Acorn Corp Cap device; it decreases LV size but does not cause constrictive physiology (22). There are no blood flow studies with the Acorn device. There is a recent report using an inert biodegradable polyester urethane cardiac patch applied to rats 2 weeks after coronary ligation where the LV cavity size does not change but fractional area change increases and compliance improves; there are no blood flow data in this report (6).

Application of a Patch as an Alternative to Direct Cell Injection

The use of a biodegradable patch that provides a support structure allowing new cells to attach and grow in a damaged heart is a possible alternative to the current approach of direct cell injection for cell-based therapy. Not only are the results from current clinical trials of cell-based therapy disappointing, the approach used in these trials is cumbersome, requiring harvesting bone marrow and a repeat cardiac catheterization with infarct artery reocclusion to reinject purified autologous mononuclear cells into the coronary arteries. Another problem is the recent report that intracoronary delivery of bone marrow cells results in damage to the coronary artery with luminal loss in the infarct related artery (20). These data suggest that we need new options for cell-based therapy for heart failure.

The translational aspect of this work is important; there is potential for clinical application of this 3DFC patch. At present there are two ongoing phase I clinical trials using the 3DFC; the first is a pilot trial in patients applying the 3DFC patch at the time of coronary artery bypass surgery when the surgeon cannot place a graft to a area of viable myocardium. This trial is designed to determine if the 3DFC increases myocardial perfusion to an area that the surgeon could not graft. While in this clinical study the 3DFC patch is placed with the chest open, two cases have been done with a minimally invasive approach using a modified video-assisted thorascopic surgery VATS procedure. The second trial is in patients getting a left ventricular assist device (LVAD). The 3DFC is applied at the time of LVAD placement and, upon LVAD removal, histology is done on the area of 3DFC placement in order to examine for evidence of angiogenesis.

Summary

We report improvements in myocardial blood flow, regional and global LV function, and partial reversal of LV remodeling using a viable three-dimensional fibroblast patch implanted in rats at the time of an acute MI. This patch provides a support structure that allows cells to grow into the damaged heart and creates new blood vessel growth, resulting in improved blood flow. With the limited success of direct cell injection into the heart, the 3DFC represents a new approach to cell-based therapy for heart failure.

Figures

Figure 1. Scanning electron micrograph of the 3DFC patch

Figure 1. Scanning electron micrograph of the 3DFC patch.

The vicryl fibers are “tube-like” structures. The fibroblasts look like irregular structures with long appendages that span from one vicryl fiber to another.

Figure 2. Three-dimensional fibroblast culture (3DFC)

Figure 2.

(A) Three-dimensional fibroblast culture (3DFC) prior to implantation; the suture in the middle of the patch is used to attach the 3DFC to the left ventricle. (B) 3DFC at the time of implantation on the infarcted left ventricle. (C) 3DFC at 3 weeks after myocardial infarction. Note that the 3DFC is well integrated and attached to the infarcted wall. (D) 3DFC in a perfused heart preparation at 3 weeks after myocardial infarction. As note above, the 3DFC is well integrated into the infarcted wall and the suture is easily visible.

Figure 3. Echocardiographic measured ejection fraction (EF)

Figure 3.

Echocardiographic measured ejection fraction (EF) in sham, myocardial infarction (MI), MI + 3DFC, MI + 3DFC/Cap (captopril), and MI + 3DFC/NV (nonviable). Note that the viable 3DFC increased the EF. The EF remained increased with the addition of captopril to the viable 3DFC; the nonviable 3DFC did not improve EF. Values are mean ± SE. Sham (N = 5); MI (N = 8); MI + 3DFC/cap (N = 10); MI + 3DFC (N = 14); MI + 3DFC (nonviable) (N = 5). *p < 0.05 sham versus all groups; **p < 0.05 MI and MI + 3DFC/NV versus MI + 3DFC/cap and MI + 3DFC.

Figure 4.

Echocardiographic measured systolic displacement of the infarcted anterior wall in sham, myocardial infarction (MI), and MI + 3DFC. Note that the 3DFC improved EF back toward the normal value. Values are mean ± SE. Sham (N = 6); MI (N = 12); MI + 3DFC (N = 15); MI + NV 3DFC (N = 12). *p < 0.05 versus MI; **p < 0.05 versus MI.

Figure 5. A. Echocardiographic measured LV end-diastolic and end-systolic diameters

Figure 5. B. Echocardiographic measured LV end-diastolic and end-systolic diameters

Figure 5.

Echocardiographic measured LV end-diastolic and end-systolic diameters in sham, myocardial infarction (MI), and MI + 3DFC. Note that both the LV end-diastolic diameter and end-systolic diameters decrease with the 3 DFC. Values are mean ± SE. Sham (N=6); MI (N=12); MI + 3DFC (N=15); MI + NV 3DFC, (N=12). *p < 0.05 versus sham; **p < 0.05 versus MI.

Figure 6. Pressure–volume (PV) loops

Figure 6.

Pressure–volume (PV) loops in sham, myocardial infarction (MI), MI + 3DFC, and MI + 3DFC/ captopril. Note that the major shift in the PV loop was with the addition of captopril where the operating LV end-diastolic volume decreased.

Figure 7.

Anterior wall myocardial blood flow in sham (N = 11), at the time of acute myocardial infarction (MI, N = 7), MI at 3 weeks (N = 4), and MI at 3 weeks with 3DFC (N = 4). Note that the 3DFC improved blood flow in the infarcted wall. Values are mean ± SE; *p < 0.05 versus baseline and MI (3w) + 3DFC.

Figure 8

Vessel density defined by Factor VIII staining. Note the increase in vessel density in the area with the 3DFC compared to the untreated myocardial infarction (MI). MI (N = 9), MI + 3DFC (N = 8). Values are mean ± SE. *p < 0.05 versus MI.

Figure 9. Histopathology

Figure 9.

Histopathology sections of Factor VIII staining in MI + 3DFC (A–C) and MI alone (4× and 40×). Note the increased in Factor VIII staining and vessel density with the 3DFC.

 

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