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Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium
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Biomaterials 26 (2005) 319–326
Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium Ju Hee Ryua,b, Il-Kwon Kimc,e, Seung-Woo Choa,b, Myeong-Chan Chod, Kyung-Kuk Hwangd, Hainan Piaod, Shuguang Piaod, Sang Hyun Lime, Yoo Sun Honge, Cha Yong Choib, Kyung Jong Yooe,*, Byung-Soo Kima,* a
Department of Chemical Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, South Korea b School of Chemical Engineering, Seoul National University, Seoul 151-742, South Korea c Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, South Korea d Department of Cardiology, College of Medicine, Chungbuk National University, South Korea e Division of Cardiovascular Surgery, Yonsei Cardiovascular Hospital and Research Institute, Yonsei University College of Medicine, Seoul 120-752, South Korea Received 12 January 2004; accepted 16 February 2004
Abstract Neovascularization may improve cardiac function and prevent further scar tissue formation in infarcted myocardium. A number of studies have demonstrated that bone marrow-derived cells have the potential to induce neovascularization in ischemic tissues. In this study, we hypothesized that implantation of bone marrow mononuclear cells (BMMNCs) using injectable fibrin matrix further enhances neovascularization in infarcted myocardium compared to BMMNC implantation without matrix. To test this hypothesis, infarction was induced in rat myocardium by cryoinjury. Three weeks later, rat BMMNCs were mixed with fibrin matrix and injected into the infarcted myocardium. Injection of either BMMNCs or medium alone into infarcted myocardium served as controls. Eight weeks after the treatments, histological analyses indicated that implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration in the infarcted myocardium compared to BMMNC implantation without matrix. Examination with fluorescence microscopy revealed that cells labeled with a fluorescent dye prior to implantation survived in the infarcted myocardium at 8 weeks of implantation. Importantly, implantation of BMMNCs using fibrin matrix resulted in much more extensive neovascularization in infarcted myocardium than BMMNC implantation without matrix. The microvessel density in infarcted myocardium was significantly higher (po0:05) when BMMNCs were implanted using fibrin matrix (350722 microvessels/ mm2) compared to BMMNC implantation without matrix (262713 microvessels/mm2) and medium injection (7679 microvessels/ mm2). In addition, average internal diameter of microvessels was significantly larger (po0:05) in BMMNC implantation with fibrin matrix group (14.671.2 mm) than BMMNC implantation without matrix group (10.270.7 mm) and medium injection group (7.370.5 mm). These results suggest that fibrin matrix could serve as a cell implantation matrix that enhances neovascularization efficacy for myocardial infarction treatment. r 2004 Elsevier Ltd. All rights reserved. Keywords: Fibrin marix; Bone marrow mononuclear cells; Myocardial infarction; Neovascularization
1. Introduction Myocardial infarction may result in left ventricle remodeling and subsequent heart failure. During the left ventricular remodeling process, injured cardiomyocytes *Corresponding authors. Tel.: +82-2-361-7280; fax: +82-2-3132992 (K.J. Yoo), Tel.: +82-2-2290-0491; fax: +82-2-2298-4101 (B.-S. Kim). E-mail addresses: [email protected] (K.J. Yoo), [email protected] (B.-S. Kim). 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.02.058
are gradually replaced by fibrous tissue [1], the initial infarct area progressively expands, and the left ventricle dilates, which may lead to heart failure [2]. An effective method to reverse myocardial remodeling is to induce neovascularization within the infarcted myocardium [3,4]. Neovascularization may occur within the infarcted myocardium even under normal circumstances without any treatment. However, this may not be sufficient to support tissue growth required for contractile compensation and to satisfy the greater demands of the hypertrophied but viable myocardium [5]. The relative
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lack of oxygen and nutrients to the hypertrophied myocardium may result in the death of myocardium. Therefore, more extensive neovascularization is required to reverse myocardial remodeling and subsequent heart failure. Neovascularization can be stimulated by angiogenic gene therapy [6], angiogenic cytokine administration [7], and transmyocardial laser revascularization [8]. However, these treatments have been applied in only a few clinical trials due to problems related to the unstable effect, the risk of systemic or local toxicity, and difficult techniques [9]. Implantation of bone marrow cells (BMCs) is attractive for the induction of neovascularization, since BMCs can differentiate into cardiomyocytes, endothelial cells and vascular smooth muscle cells [10] and secrete various angiogenic growth factors [11]. In addition, implantation of autologous BMCs avoids immunorejection. In clinical trials, implantation of bone marrow mononuclear cells (BMMNCs) into infarcted myocardium demonstrated myocardial regeneration, neovascularization, and treatment safety [12]. In the present study, we tested the hypothesis that implantation of BMMNCs using a cell implantation matrix enhances neovascularization in the infarcted myocardium compared to BMMNC implantation without matrix. The rationale of this hypothesis is that cell adhesion to matrix may be necessary for the differentiation of BMCs into somatic mesenchymal cells [13] and for the survival of the differentiated adherent cells, including endothelial cells [14]. Fibrin matrix was utilized as a cell implantation matrix in this study because fibrin is easily injectable and autologous fibrin avoids the potential risk of foreign body reactions. Rat BMMNCs were mixed with fibrin matrix and injected into infarcted myocardium in rat myocardium 3 weeks after cryoinjury. Injection of either BMMNCs or medium alone into infarcted myocardium served as controls. Neovascularization in each group was evaluated by determining the density and average internal diameter of microvessels in the infarcted myocardium 8 weeks after the treatments. Tissue regeneration and implanted cell survival in the infarcted myocardium were also examined.
2. Materials and methods 2.1. Rat myocardial infarction model Sprague-Dawley rats (200–250 g, SLC, Tokyo, Japan) were anesthetized with an intramuscular administration of ketamin hydrochloride (90 mg/kg) and xylazine hydrochloride (5 mg/kg). The anesthetized rats were incubated and placed on a ventilator (model 683, Harvard Apparatus, South Natick, MA, USA). The
rat heart was exposed through a 2-cm left lateral thoracotomy. Cryoinjury was made with a metal probe (8 mm in diameter) cooled by immersion in liquid nitrogen. The cooled metal probe was applied to the left ventricle free wall for 10 s twice, afterwards for 60 s six times. The muscle layer and the skin incision were closed with sutures. All care and handling of animals were performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH publication 85–23, revised 1985). 2.2. BMMNC isolation BMCs were flushed from the femurs and tibias of Sprague-Dawley rats (200–250 g) into culture medium (Medium 199; Gibco BRL, Gaithersburg, MD, USA). The cell suspension was loaded on Ficoll-Paque density gradient (specific gravity=1.077, Amersham Biosciences, Arlington Heights, IL, USA), and centrifuged for 20 min at 230 g. BMMNCs were isolated from the layer between the Ficoll-Paque reagent and blood plasma, and washed three times in phosphate-buffered saline (PBS; Sigma, St. Louis, MO, USA). 2.3. BMMNC labeling and detection Prior to implantation, BMMNCs were labeled with the fluorescent probe Cell TrackerTM chloromethyl-1,1dioactadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (CM-DiI; Molecular Probes, Eugene, OR, USA) that incorporates into cell membranes. BMMNCs were incubated serially in Hank’s balanced salt solution (Sigma) containing 1 mg/ml CM-DiI dye at 37 C for 5 min and at 4 C for 15 min. The labeled cells were washed three times in PBS and used immediately for implantation. Eight weeks after implantation, fluorescently labeled BMMNCs were detected using a fluorescence microscope (Eclipse E800, Nikon, Tokyo, Japan). 2.4. Fibrin matrix preparation Fibrin matrix was prepared from a commercially available fibrin gel kit (Greenplasts, GreencrossPD Co., Yongin, Korea). Plasminogen-free fibrinogen (100 mg) and fibrin-stabilizing factor XIII (66 units) were dissolved in 1 ml of plasmin inhibitor aprotinin solution (1100 kIU/ ml) for fibrinogen solution. Thrombin (500 IU) was dissolved in 1 ml of calcium chloride solution (5.9 mg/ml) for thrombin solution. The fibrinogen solution and thrombin solution containing cells were mixed at a 1:1 volume ratio and injected into infarcted myocardium. 2.5. Cell implantation Thrombin solution (100 ml) containing BMMNCs (2 107 cells) and fibrinogen solution (100 ml) were
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injected into infarcted myocardium through a device designed for simultaneous injection of the fibrinogen and thrombin solutions. Two injections (100 ml per injection) were administered in each infarcted myocardium. Injection of either BMMNCs suspended in medium (Medium 199) or medium alone into infarcted myocardium served as controls. Injection of BMMNCs suspended in medium was performed at the same cell concentration and volume as those of injection of BMMNCs using fibrin matrix.
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2.8. Statistical analysis Data were obtained from three animals per group and expressed as the mean7standard deviation. Statistical analysis was performed using unpaired Student’s t-test (InStat, GraphPad Software Inc., San Diego, CA, USA). A value of po0:05 was considered to be statistically significant.
3. Results 2.6. Histological and immunohistochemical analyses Eight weeks after the treatments, the whole hearts were retrieved and fixed in 10%(v/v) buffered formalin solution, dehydrated with a graded ethanol series, and embedded in paraffin. The specimens at the implantation sites were cut into 4 mm-thick sections and stained with hematoxylin and eosin (H&E). Collagen in the tissue sections was stained using the Masson’s trichrome method. For immunohistochemical analyses, 4 mm-thick sections were stained using antibodies against a-smooth muscle actin (a-SMA; DAKO, Carpenteria, CA, USA). The staining signals were developed with an avidinperoxidase system (Vectastains Elite ABC kit, Vector Laboratories, Burlingame, CA, USA). Gill’s hematoxylin (Sigma) was used for counterstaining.
2.7. Evaluation of microvessels in infarcted myocardium The density and internal diameter of microvessels were measured from the tissue sections stained with H&E. The density and internal diameter of microvessels were determined by analyzing at least 100 microvessels from a minimum of 10 individual photomicrograph images.
A rat myocardial infarction model was created by cryoinjury. Three weeks after cryoinjury, histological analyses indicated that the wall of infarcted myocardium became thin in all animals (Fig. 1A), which was caused by the necrosis of the infarcted tissues. No viable cells and vessels were observed in the infarcted myocardium (Fig. 1B). Twenty million BMMNCs were mixed with fibrin matrix and injected into infarcted myocardium (Fig. 2). Polymerization of fibrinogen and thrombin solutions resulted in fibrin gel formation after the injection. Fibrin matrix was easily injected without needle obstruction. Eight weeks after the treatment, H&E staining indicated that implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration in the infarcted myocardium, compared to BMMNC implantation without matrix (Figs. 3A, B and 4). However, no typical structure of cardiac muscle was observed in the infarction sites in both groups of BMMNC implantation with and without fibrin matrix. No significant inflammatory and immune reaction was observed in the infarction sites in both groups. Fibrin matrix disappeared completely 8 weeks after treatment. Little change was observed in the infarcted myocardium in the medium injection group between before and 8 weeks after medium injection (Figs. 1B and 3C).
Fig. 1. Histological sections (H&E staining) of whole heart with myocardial infarction 3 weeks after cryoinjury. (A) The myocardium wall became thin in the infarction sites (arrows). (B) No viable cells and vessels were observed in the infarction site. The scale bars indicate (A) 2 mm and (B) 100 mm.
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Masson’s trichrome staining indicated that a larger amount of viable tissues and a smaller amount of fibrous tissues were present in the group of BMMNC implantation with fibrin matrix compared to the group of BMMNC implantation without matrix (Fig. 4). In the Masson’s trichrome staining of infarcted myocardium, infarction tissue replaced with fibroblasts and collagen
Fig. 2. Photograph of cell injection into infarcted myocardium. Thrombin solution containing BMMNCs and fibrinogen solution were injected into infarcted myocardium through a device designed for simultaneous injection of the fibrinogen and thrombin solutions.
appears blue and viable myocardium appears red [15]. The viable tissues in the infarction sites were likely formed by the implanted cells. Examination with fluorescence microscopy showed that cells labeled with a fluorescent dye prior to implantation were detected in the infarction sites in both groups of BMMNC implantation with and without fibrin matrix (Fig. 5), which suggests the implanted cells survived in the infarction sites after implantation. Implantation of BMMNCs using fibrin matrix resulted in more extensive neovascularization in infarcted myocardium than BMMNC implantation without matrix. To evaluate mature vessel formation in the infarction sites, we stained tissue sections with antibodies against a-SMA, which is expressed in smooth muscle cells in mature blood vessels [16]. A larger number of microvessels were positively stained and larger vessels were formed in the group of BMMNC implantation with fibrin matrix than groups of BMMNC implantation without matrix and medium injection (Fig. 6). This result was confirmed by quantification of the density and internal diameter of microvessels in the infarction sites stained with H&E. The microvessel density in infarction sites was significantly higher (po0:05) when BMMNCs were implanted with fibrin matrix (350722 microvessels/mm2) compared to BMMNC implantation without matrix
Fig. 3. H&E staining of the infarcted myocardium 8 weeks after the treatments. Tissue regeneration in infarction sites was more extensive in (A) the group of BMMNC implantation using fibrin matrix than (B) BMMNC implantation without matrix group and (C) medium injection group. The scale bars indicate 100 mm.
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Fig. 4. Masson’s trichrome staining of the infarcted myocardium 8 weeks after the treatments. The arrows indicate infarction sites. (A,B,C) BMMNC implantation with fibrin matrix (scale bar=(A, B) 2 mm, and (C) 100 mm). (D,E,F) BMMNC implantation without matrix (scale bar=(D, E) 2 mm, and (F) 100 mm). A larger amount of viable tissues (appearing red color) and a smaller amount of fibrous tissues (appearing blue color) were present in the group of BMMNC implantation with fibrin matrix than the group of BMMNC implantation without matrix.
Fig. 5. Detection of fluorescently labeled BMMNCs 8 weeks after implantation. Examination with fluorescence microscopy revealed that cells labeled with a fluorescent dye (CM-DiI) prior to implantation were still present in infarction sites in both groups of BMMNC implantation (A) with and (B) without fibrin matrix. The scale bars indicate 50 mm.
(262713 microvessels/mm2) and medium injection (7679 microvessels/mm2) (Fig. 7). In addition, the average internal diameter of microvessels was significantly larger (po0:05) in BMMNC implantation with fibrin matrix group (14.671.2 mm) than BMMNC implantation without matrix group (10.270.7 mm) and medium injection group (7.370.5 mm) (Fig. 8).
4. Discussion A number of studies have demonstrated that BMCs contain endothelial precursors [17] and that BMC
implantation induces neovascularization in ischemic tissues both experimentally [18–21] and clinically [22,23]. In this study, we tested the hypothesis that BMC implantation using a cell implantation matrix enhances neovascularization compared to BMC implantation without matrix. Rat BMMNCs mixed with fibrin matrix were implanted into rat infarcted myocardium. Eight weeks after the treatment, implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration and neovascularization compared to BMMNC implantation without matrix. BMC implantation could be an effective method to induce neovascularization in ischemic tissues. Gene therapy has been proposed as a potential method to induce therapeutic angiogenesis in myocardial infarction sites. Experimental data indicate that adenoviral and plasmid vectors encoding various angiogenic cytokines were able to induce significant angiogenesis in vitro and in animal models of myocardial infarction [24]. However, the morbidity and mortality accompanying gene delivery technique are major obstacles to its use [25]. BMCs contain endothelial precursors, which could differentiate into endothelial cells [10,17]. The potential of BMCs to induce neovascularization in ischemic tissues has been demonstrated in a number of studies [9,23,26]. BMC implantation could be a safe method because it avoids immunorejection when autologous cells are used. In addition, BMC implantation could regenerate cardiac muscle as well as blood vessels in infarcted myocardium, which is critical for heart function recovery [10].
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Fig. 6. Immunohistochemical staining for a-SMA of the myocardial infarction sites 8 weeks after the treatments. A larger number of microvessels were positively stained and larger vessels were formed in (A) BMMNC implantation with fibrin matrix group than (B) BMMNC implantation without matrix group and (C) medium injection group. The scale bars indicate 100 mm.
∗
400
∗
300 Microvessels/mm2
∗
200
100
0
Medium only
BMMNCs only
BMMNCs + Fibrin matrix
Fig. 7. Microvessel density in the myocardial infarction sites 8 weeks after the treatments. The microvessel density in infarction sites was significantly higher (po0:05) when BMMNCs were implanted using fibrin matrix (350722 microvessels/mm2) compared to BMMNC implantation without matrix (262713 microvessels/mm2) and medium injection (7679 microvessels/mm2). Asterisks indicate po0:05:
Implantation of BMMNCs using fibrin matrix resulted in more extensive neovascularization in infarcted myocardium than BMMNC implantation with-
out matrix. Neovascularization enhanced in the group of BMMNCs implanted with fibrin matrix was likely due to prevention of anoikis by BMMNC-fibrin matrix interactions. Anoikis is defined as programmed cell death induced by the loss of cell–matrix interactions [27]. Cell adhesion to matrix may be necessary for the differentiation of bone marrow stromal cells into somatic mesenchymal cells [28] and for the survival of the differentiated adherent cells, including endothelial cells [14]. Interaction between implanted BMMNCs and fibrin matrix could enhance the differentiation of the BMMNCs into endothelial cells and vascular smooth muscle cells and the survival of the differentiated cells, avoiding anoikis. Fibrin matrix has several advantages as a cell implantation matrix for cardiovascular tissue engineering. Fibrin matrix has gained FDA approval for human use in a variety of applications, including sealant [29]. In addition, fibrin matrix has been used for regeneration of skin [30] and cartilage [31], and showed successful results. Unlike xenogenic gelatin and collagen, which may induce intense inflammatory responses, fibrin can avoid the potential risk of a foreign body reaction because it can be produced from patient’s own blood [32]. Fibrin matrix could be injected endoscopically
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through a catheter, which could obviate invasive open surgery for myocardial infarction treatment. Such treatment can shorten the surgical operation time and recovery time, and reduce patients’ pain. Further investigation would be necessary to evaluate the clinical potential of this method. In this study, implantation of BMMNCs using fibrin matrix enhanced neovascularization, but did not regenerate myocardium in the infarction sites. This is consistent with the result of a previous study. Homano and colleagues reported that BMC implantation into canine infarcted myocardium improved local wall thickening, presumably due to the angiogenesis induced by the implantation, but did not regenerate myocardium [9]. Implantation of sorted BMCs [10], mobilization of primitive BMCs by cytokines [33], and implantation of 5-azacytidine-treated BMCs [20] could be considered to induce both myocardium regeneration and neovascularization in infarcted myocardium. In addition, cardiac function tests need to be performed to assess the cardiac function recovery of treated animals.
5. Conclusion Implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration and neovascularization in infarcted myocardium compared to BMMNC implantation without matrix. The results of this study suggest that fibrin matrix could serve as a cell implantation matrix that enhances neovascularization efficacy of the cell therapy for myocardial infarction treatment. The use of fibrin matrix for BMMNC implantation could be also applied for limb ischemia therapy that requires neovascularization.
Acknowledgements This study was supported by a Grant (No. 02-PJ10PG8-EC01-0016) of the Korea Health 21 R&D Project, the ministry of health & welfare, Republic of Korea.
References
Fig. 8. Distributions of internal diameter of microvessels in the myocardial infarction sites 8 weeks after the treatments. The average internal diameter of microvessels was significantly larger (po0:05) in (A) BMMNC implantation with fibrin matrix group (14.671.2 mm) than (B) BMMNC implantation without matrix group (10.270.7 mm) and (C) medium injection group (7.370.5 mm).
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Biomaterials 26 (2005) 319–326
Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium Ju Hee Ryua,b, Il-Kwon Kimc,e, Seung-Woo Choa,b, Myeong-Chan Chod, Kyung-Kuk Hwangd, Hainan Piaod, Shuguang Piaod, Sang Hyun Lime, Yoo Sun Honge, Cha Yong Choib, Kyung Jong Yooe,*, Byung-Soo Kima,* a
Department of Chemical Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, South Korea b School of Chemical Engineering, Seoul National University, Seoul 151-742, South Korea c Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, South Korea d Department of Cardiology, College of Medicine, Chungbuk National University, South Korea e Division of Cardiovascular Surgery, Yonsei Cardiovascular Hospital and Research Institute, Yonsei University College of Medicine, Seoul 120-752, South Korea Received 12 January 2004; accepted 16 February 2004
Abstract Neovascularization may improve cardiac function and prevent further scar tissue formation in infarcted myocardium. A number of studies have demonstrated that bone marrow-derived cells have the potential to induce neovascularization in ischemic tissues. In this study, we hypothesized that implantation of bone marrow mononuclear cells (BMMNCs) using injectable fibrin matrix further enhances neovascularization in infarcted myocardium compared to BMMNC implantation without matrix. To test this hypothesis, infarction was induced in rat myocardium by cryoinjury. Three weeks later, rat BMMNCs were mixed with fibrin matrix and injected into the infarcted myocardium. Injection of either BMMNCs or medium alone into infarcted myocardium served as controls. Eight weeks after the treatments, histological analyses indicated that implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration in the infarcted myocardium compared to BMMNC implantation without matrix. Examination with fluorescence microscopy revealed that cells labeled with a fluorescent dye prior to implantation survived in the infarcted myocardium at 8 weeks of implantation. Importantly, implantation of BMMNCs using fibrin matrix resulted in much more extensive neovascularization in infarcted myocardium than BMMNC implantation without matrix. The microvessel density in infarcted myocardium was significantly higher (po0:05) when BMMNCs were implanted using fibrin matrix (350722 microvessels/ mm2) compared to BMMNC implantation without matrix (262713 microvessels/mm2) and medium injection (7679 microvessels/ mm2). In addition, average internal diameter of microvessels was significantly larger (po0:05) in BMMNC implantation with fibrin matrix group (14.671.2 mm) than BMMNC implantation without matrix group (10.270.7 mm) and medium injection group (7.370.5 mm). These results suggest that fibrin matrix could serve as a cell implantation matrix that enhances neovascularization efficacy for myocardial infarction treatment. r 2004 Elsevier Ltd. All rights reserved. Keywords: Fibrin marix; Bone marrow mononuclear cells; Myocardial infarction; Neovascularization
1. Introduction Myocardial infarction may result in left ventricle remodeling and subsequent heart failure. During the left ventricular remodeling process, injured cardiomyocytes *Corresponding authors. Tel.: +82-2-361-7280; fax: +82-2-3132992 (K.J. Yoo), Tel.: +82-2-2290-0491; fax: +82-2-2298-4101 (B.-S. Kim). E-mail addresses: [email protected] (K.J. Yoo), [email protected] (B.-S. Kim). 0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.02.058
are gradually replaced by fibrous tissue [1], the initial infarct area progressively expands, and the left ventricle dilates, which may lead to heart failure [2]. An effective method to reverse myocardial remodeling is to induce neovascularization within the infarcted myocardium [3,4]. Neovascularization may occur within the infarcted myocardium even under normal circumstances without any treatment. However, this may not be sufficient to support tissue growth required for contractile compensation and to satisfy the greater demands of the hypertrophied but viable myocardium [5]. The relative
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lack of oxygen and nutrients to the hypertrophied myocardium may result in the death of myocardium. Therefore, more extensive neovascularization is required to reverse myocardial remodeling and subsequent heart failure. Neovascularization can be stimulated by angiogenic gene therapy [6], angiogenic cytokine administration [7], and transmyocardial laser revascularization [8]. However, these treatments have been applied in only a few clinical trials due to problems related to the unstable effect, the risk of systemic or local toxicity, and difficult techniques [9]. Implantation of bone marrow cells (BMCs) is attractive for the induction of neovascularization, since BMCs can differentiate into cardiomyocytes, endothelial cells and vascular smooth muscle cells [10] and secrete various angiogenic growth factors [11]. In addition, implantation of autologous BMCs avoids immunorejection. In clinical trials, implantation of bone marrow mononuclear cells (BMMNCs) into infarcted myocardium demonstrated myocardial regeneration, neovascularization, and treatment safety [12]. In the present study, we tested the hypothesis that implantation of BMMNCs using a cell implantation matrix enhances neovascularization in the infarcted myocardium compared to BMMNC implantation without matrix. The rationale of this hypothesis is that cell adhesion to matrix may be necessary for the differentiation of BMCs into somatic mesenchymal cells [13] and for the survival of the differentiated adherent cells, including endothelial cells [14]. Fibrin matrix was utilized as a cell implantation matrix in this study because fibrin is easily injectable and autologous fibrin avoids the potential risk of foreign body reactions. Rat BMMNCs were mixed with fibrin matrix and injected into infarcted myocardium in rat myocardium 3 weeks after cryoinjury. Injection of either BMMNCs or medium alone into infarcted myocardium served as controls. Neovascularization in each group was evaluated by determining the density and average internal diameter of microvessels in the infarcted myocardium 8 weeks after the treatments. Tissue regeneration and implanted cell survival in the infarcted myocardium were also examined.
2. Materials and methods 2.1. Rat myocardial infarction model Sprague-Dawley rats (200–250 g, SLC, Tokyo, Japan) were anesthetized with an intramuscular administration of ketamin hydrochloride (90 mg/kg) and xylazine hydrochloride (5 mg/kg). The anesthetized rats were incubated and placed on a ventilator (model 683, Harvard Apparatus, South Natick, MA, USA). The
rat heart was exposed through a 2-cm left lateral thoracotomy. Cryoinjury was made with a metal probe (8 mm in diameter) cooled by immersion in liquid nitrogen. The cooled metal probe was applied to the left ventricle free wall for 10 s twice, afterwards for 60 s six times. The muscle layer and the skin incision were closed with sutures. All care and handling of animals were performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH publication 85–23, revised 1985). 2.2. BMMNC isolation BMCs were flushed from the femurs and tibias of Sprague-Dawley rats (200–250 g) into culture medium (Medium 199; Gibco BRL, Gaithersburg, MD, USA). The cell suspension was loaded on Ficoll-Paque density gradient (specific gravity=1.077, Amersham Biosciences, Arlington Heights, IL, USA), and centrifuged for 20 min at 230 g. BMMNCs were isolated from the layer between the Ficoll-Paque reagent and blood plasma, and washed three times in phosphate-buffered saline (PBS; Sigma, St. Louis, MO, USA). 2.3. BMMNC labeling and detection Prior to implantation, BMMNCs were labeled with the fluorescent probe Cell TrackerTM chloromethyl-1,1dioactadecyl-3,3,30 ,30 -tetramethylindocarbocyanine perchlorate (CM-DiI; Molecular Probes, Eugene, OR, USA) that incorporates into cell membranes. BMMNCs were incubated serially in Hank’s balanced salt solution (Sigma) containing 1 mg/ml CM-DiI dye at 37 C for 5 min and at 4 C for 15 min. The labeled cells were washed three times in PBS and used immediately for implantation. Eight weeks after implantation, fluorescently labeled BMMNCs were detected using a fluorescence microscope (Eclipse E800, Nikon, Tokyo, Japan). 2.4. Fibrin matrix preparation Fibrin matrix was prepared from a commercially available fibrin gel kit (Greenplasts, GreencrossPD Co., Yongin, Korea). Plasminogen-free fibrinogen (100 mg) and fibrin-stabilizing factor XIII (66 units) were dissolved in 1 ml of plasmin inhibitor aprotinin solution (1100 kIU/ ml) for fibrinogen solution. Thrombin (500 IU) was dissolved in 1 ml of calcium chloride solution (5.9 mg/ml) for thrombin solution. The fibrinogen solution and thrombin solution containing cells were mixed at a 1:1 volume ratio and injected into infarcted myocardium. 2.5. Cell implantation Thrombin solution (100 ml) containing BMMNCs (2 107 cells) and fibrinogen solution (100 ml) were
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injected into infarcted myocardium through a device designed for simultaneous injection of the fibrinogen and thrombin solutions. Two injections (100 ml per injection) were administered in each infarcted myocardium. Injection of either BMMNCs suspended in medium (Medium 199) or medium alone into infarcted myocardium served as controls. Injection of BMMNCs suspended in medium was performed at the same cell concentration and volume as those of injection of BMMNCs using fibrin matrix.
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2.8. Statistical analysis Data were obtained from three animals per group and expressed as the mean7standard deviation. Statistical analysis was performed using unpaired Student’s t-test (InStat, GraphPad Software Inc., San Diego, CA, USA). A value of po0:05 was considered to be statistically significant.
3. Results 2.6. Histological and immunohistochemical analyses Eight weeks after the treatments, the whole hearts were retrieved and fixed in 10%(v/v) buffered formalin solution, dehydrated with a graded ethanol series, and embedded in paraffin. The specimens at the implantation sites were cut into 4 mm-thick sections and stained with hematoxylin and eosin (H&E). Collagen in the tissue sections was stained using the Masson’s trichrome method. For immunohistochemical analyses, 4 mm-thick sections were stained using antibodies against a-smooth muscle actin (a-SMA; DAKO, Carpenteria, CA, USA). The staining signals were developed with an avidinperoxidase system (Vectastains Elite ABC kit, Vector Laboratories, Burlingame, CA, USA). Gill’s hematoxylin (Sigma) was used for counterstaining.
2.7. Evaluation of microvessels in infarcted myocardium The density and internal diameter of microvessels were measured from the tissue sections stained with H&E. The density and internal diameter of microvessels were determined by analyzing at least 100 microvessels from a minimum of 10 individual photomicrograph images.
A rat myocardial infarction model was created by cryoinjury. Three weeks after cryoinjury, histological analyses indicated that the wall of infarcted myocardium became thin in all animals (Fig. 1A), which was caused by the necrosis of the infarcted tissues. No viable cells and vessels were observed in the infarcted myocardium (Fig. 1B). Twenty million BMMNCs were mixed with fibrin matrix and injected into infarcted myocardium (Fig. 2). Polymerization of fibrinogen and thrombin solutions resulted in fibrin gel formation after the injection. Fibrin matrix was easily injected without needle obstruction. Eight weeks after the treatment, H&E staining indicated that implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration in the infarcted myocardium, compared to BMMNC implantation without matrix (Figs. 3A, B and 4). However, no typical structure of cardiac muscle was observed in the infarction sites in both groups of BMMNC implantation with and without fibrin matrix. No significant inflammatory and immune reaction was observed in the infarction sites in both groups. Fibrin matrix disappeared completely 8 weeks after treatment. Little change was observed in the infarcted myocardium in the medium injection group between before and 8 weeks after medium injection (Figs. 1B and 3C).
Fig. 1. Histological sections (H&E staining) of whole heart with myocardial infarction 3 weeks after cryoinjury. (A) The myocardium wall became thin in the infarction sites (arrows). (B) No viable cells and vessels were observed in the infarction site. The scale bars indicate (A) 2 mm and (B) 100 mm.
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Masson’s trichrome staining indicated that a larger amount of viable tissues and a smaller amount of fibrous tissues were present in the group of BMMNC implantation with fibrin matrix compared to the group of BMMNC implantation without matrix (Fig. 4). In the Masson’s trichrome staining of infarcted myocardium, infarction tissue replaced with fibroblasts and collagen
Fig. 2. Photograph of cell injection into infarcted myocardium. Thrombin solution containing BMMNCs and fibrinogen solution were injected into infarcted myocardium through a device designed for simultaneous injection of the fibrinogen and thrombin solutions.
appears blue and viable myocardium appears red [15]. The viable tissues in the infarction sites were likely formed by the implanted cells. Examination with fluorescence microscopy showed that cells labeled with a fluorescent dye prior to implantation were detected in the infarction sites in both groups of BMMNC implantation with and without fibrin matrix (Fig. 5), which suggests the implanted cells survived in the infarction sites after implantation. Implantation of BMMNCs using fibrin matrix resulted in more extensive neovascularization in infarcted myocardium than BMMNC implantation without matrix. To evaluate mature vessel formation in the infarction sites, we stained tissue sections with antibodies against a-SMA, which is expressed in smooth muscle cells in mature blood vessels [16]. A larger number of microvessels were positively stained and larger vessels were formed in the group of BMMNC implantation with fibrin matrix than groups of BMMNC implantation without matrix and medium injection (Fig. 6). This result was confirmed by quantification of the density and internal diameter of microvessels in the infarction sites stained with H&E. The microvessel density in infarction sites was significantly higher (po0:05) when BMMNCs were implanted with fibrin matrix (350722 microvessels/mm2) compared to BMMNC implantation without matrix
Fig. 3. H&E staining of the infarcted myocardium 8 weeks after the treatments. Tissue regeneration in infarction sites was more extensive in (A) the group of BMMNC implantation using fibrin matrix than (B) BMMNC implantation without matrix group and (C) medium injection group. The scale bars indicate 100 mm.
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Fig. 4. Masson’s trichrome staining of the infarcted myocardium 8 weeks after the treatments. The arrows indicate infarction sites. (A,B,C) BMMNC implantation with fibrin matrix (scale bar=(A, B) 2 mm, and (C) 100 mm). (D,E,F) BMMNC implantation without matrix (scale bar=(D, E) 2 mm, and (F) 100 mm). A larger amount of viable tissues (appearing red color) and a smaller amount of fibrous tissues (appearing blue color) were present in the group of BMMNC implantation with fibrin matrix than the group of BMMNC implantation without matrix.
Fig. 5. Detection of fluorescently labeled BMMNCs 8 weeks after implantation. Examination with fluorescence microscopy revealed that cells labeled with a fluorescent dye (CM-DiI) prior to implantation were still present in infarction sites in both groups of BMMNC implantation (A) with and (B) without fibrin matrix. The scale bars indicate 50 mm.
(262713 microvessels/mm2) and medium injection (7679 microvessels/mm2) (Fig. 7). In addition, the average internal diameter of microvessels was significantly larger (po0:05) in BMMNC implantation with fibrin matrix group (14.671.2 mm) than BMMNC implantation without matrix group (10.270.7 mm) and medium injection group (7.370.5 mm) (Fig. 8).
4. Discussion A number of studies have demonstrated that BMCs contain endothelial precursors [17] and that BMC
implantation induces neovascularization in ischemic tissues both experimentally [18–21] and clinically [22,23]. In this study, we tested the hypothesis that BMC implantation using a cell implantation matrix enhances neovascularization compared to BMC implantation without matrix. Rat BMMNCs mixed with fibrin matrix were implanted into rat infarcted myocardium. Eight weeks after the treatment, implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration and neovascularization compared to BMMNC implantation without matrix. BMC implantation could be an effective method to induce neovascularization in ischemic tissues. Gene therapy has been proposed as a potential method to induce therapeutic angiogenesis in myocardial infarction sites. Experimental data indicate that adenoviral and plasmid vectors encoding various angiogenic cytokines were able to induce significant angiogenesis in vitro and in animal models of myocardial infarction [24]. However, the morbidity and mortality accompanying gene delivery technique are major obstacles to its use [25]. BMCs contain endothelial precursors, which could differentiate into endothelial cells [10,17]. The potential of BMCs to induce neovascularization in ischemic tissues has been demonstrated in a number of studies [9,23,26]. BMC implantation could be a safe method because it avoids immunorejection when autologous cells are used. In addition, BMC implantation could regenerate cardiac muscle as well as blood vessels in infarcted myocardium, which is critical for heart function recovery [10].
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Fig. 6. Immunohistochemical staining for a-SMA of the myocardial infarction sites 8 weeks after the treatments. A larger number of microvessels were positively stained and larger vessels were formed in (A) BMMNC implantation with fibrin matrix group than (B) BMMNC implantation without matrix group and (C) medium injection group. The scale bars indicate 100 mm.
∗
400
∗
300 Microvessels/mm2
∗
200
100
0
Medium only
BMMNCs only
BMMNCs + Fibrin matrix
Fig. 7. Microvessel density in the myocardial infarction sites 8 weeks after the treatments. The microvessel density in infarction sites was significantly higher (po0:05) when BMMNCs were implanted using fibrin matrix (350722 microvessels/mm2) compared to BMMNC implantation without matrix (262713 microvessels/mm2) and medium injection (7679 microvessels/mm2). Asterisks indicate po0:05:
Implantation of BMMNCs using fibrin matrix resulted in more extensive neovascularization in infarcted myocardium than BMMNC implantation with-
out matrix. Neovascularization enhanced in the group of BMMNCs implanted with fibrin matrix was likely due to prevention of anoikis by BMMNC-fibrin matrix interactions. Anoikis is defined as programmed cell death induced by the loss of cell–matrix interactions [27]. Cell adhesion to matrix may be necessary for the differentiation of bone marrow stromal cells into somatic mesenchymal cells [28] and for the survival of the differentiated adherent cells, including endothelial cells [14]. Interaction between implanted BMMNCs and fibrin matrix could enhance the differentiation of the BMMNCs into endothelial cells and vascular smooth muscle cells and the survival of the differentiated cells, avoiding anoikis. Fibrin matrix has several advantages as a cell implantation matrix for cardiovascular tissue engineering. Fibrin matrix has gained FDA approval for human use in a variety of applications, including sealant [29]. In addition, fibrin matrix has been used for regeneration of skin [30] and cartilage [31], and showed successful results. Unlike xenogenic gelatin and collagen, which may induce intense inflammatory responses, fibrin can avoid the potential risk of a foreign body reaction because it can be produced from patient’s own blood [32]. Fibrin matrix could be injected endoscopically
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through a catheter, which could obviate invasive open surgery for myocardial infarction treatment. Such treatment can shorten the surgical operation time and recovery time, and reduce patients’ pain. Further investigation would be necessary to evaluate the clinical potential of this method. In this study, implantation of BMMNCs using fibrin matrix enhanced neovascularization, but did not regenerate myocardium in the infarction sites. This is consistent with the result of a previous study. Homano and colleagues reported that BMC implantation into canine infarcted myocardium improved local wall thickening, presumably due to the angiogenesis induced by the implantation, but did not regenerate myocardium [9]. Implantation of sorted BMCs [10], mobilization of primitive BMCs by cytokines [33], and implantation of 5-azacytidine-treated BMCs [20] could be considered to induce both myocardium regeneration and neovascularization in infarcted myocardium. In addition, cardiac function tests need to be performed to assess the cardiac function recovery of treated animals.
5. Conclusion Implantation of BMMNCs using fibrin matrix resulted in more extensive tissue regeneration and neovascularization in infarcted myocardium compared to BMMNC implantation without matrix. The results of this study suggest that fibrin matrix could serve as a cell implantation matrix that enhances neovascularization efficacy of the cell therapy for myocardial infarction treatment. The use of fibrin matrix for BMMNC implantation could be also applied for limb ischemia therapy that requires neovascularization.
Acknowledgements This study was supported by a Grant (No. 02-PJ10PG8-EC01-0016) of the Korea Health 21 R&D Project, the ministry of health & welfare, Republic of Korea.
References
Fig. 8. Distributions of internal diameter of microvessels in the myocardial infarction sites 8 weeks after the treatments. The average internal diameter of microvessels was significantly larger (po0:05) in (A) BMMNC implantation with fibrin matrix group (14.671.2 mm) than (B) BMMNC implantation without matrix group (10.270.7 mm) and (C) medium injection group (7.370.5 mm).
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