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Porous acellular bovine pericardia seeded with mesenchymal stem cells as a patch to repair a myocardial defect in a syngeneic rat model Hao-Ji Weia, Sung-Ching Chenb, Yen Changa, Shiaw-Min Hwangc, Wei-Wen Lind, Po-Hong Laib, Huihua Kenny Chiange, Lee-Feng Hsuc, Hang-Hsing Yangb, Hsing-Wen Sungb, a
Division of Cardiovascular Surgery, Veterans General Hospital-Taichung, and College of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC b Department of Chemical Engineering, Bioengineering Program, National Tsing Hua University, Hsinchu, Taiwan 30013, ROC c Food Industry Research and Development Institute, Culture Collection and Research Center, Hsinchu, Taiwan, ROC d Division of Cardiology, Veterans General Hospital-Taichung, and Department of Life Science, Tunghai University, Taichung, Taiwan, ROC e Institute of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan, ROC Received 28 March 2006; accepted 29 June 2006 Available online 17 July 2006
Abstract A patch is often mandatory to repair myocardial defects; however, currently available patches lack the possibility of regeneration. To overcome this limitation, a porous acellular bovine pericardium seeded with BrdU-labeled mesenchymal stem cells (MSCs) was prepared (the MSC patch) to repair a surgically created myocardial defect in the right ventricle of a syngeneic rat model. The bovine pericardium before cell extraction was used as a control (the Control patch). The implanted samples were retrieved at 4- and 12-week postoperatively (n ¼ 5 per group at each time point). At retrieval, no aneurysmal dilation of the implanted patches was seen for both studied groups. No apparent tissue adhesion was observed for the MSC patch throughout the entire course of the study, while for the Control patch, two out of the five studied animals at 12-week postoperatively had a filmy adhesion to the chest wall. On the inner (endocardial) surface, intimal thickening was observed for both studied groups; however, no thrombus formation was found. Intact layers of endothelial and mesothelial cells were identified on the inner and outer (epicardial) surfaces of the MSC patch. Smooth muscle cells together with neomuscle fibers, neo-glycosaminoglycans and neo-capillaries were observed within the pores of the MSC patch. Some cardiomyocytes, which stained positively for BrdU and a-sacromeric actin, were observed in the MSC patch, indicating that the implanted MSCs can engraft and differentiate into cardiomyocytes. Additionally, a normality of the local electrograms on the epicardial surface of the MSC patch was observed. In contrast, no apparent tissue regeneration was observed for the Control patch throughout the entire course of the study, while only abnormal electrogram signals were seen on its epicardial surface. In conclusion, the MSC patch may preserve the structure of the ventricular wall while providing the potential for myocardial tissue regeneration. r 2006 Elsevier Ltd. All rights reserved. Keywords: Mesenchymal stem cells; Porous acellular bovine pericardium; Cardiomyocytes; Myocardial tissue regeneration; Angiogenesis
1. Introduction Myocardial infarction progresses from the acute death of cardiomyocytes and the infiltration of inflammatory cells into granulation, followed by scars [1]. This results in a progressive loss of functional myocardium and a successive reduction in cardiac performance [2]. A patch is often Corresponding author. Fax: +886 3 572 6832.
E-mail address:
[email protected] (H.-W. Sung). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.06.022
mandatory to repair failing myocardium [3]. Currently, the patches used clinically are made of Dacron polyester fabric, expanded polytetrafluoroethylene (e-PTFE), glutaraldehyde-treated bovine pericardia or cryopreserved homografts [4]. However, their long-term results are compromised by material-related failures because these materials are not viable [4]. In our previous study, a cell extraction process developed by Courtman et al. [5] was adopted to remove the cellular components of bovine pericardia. It was found that
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the acellular bovine pericardial tissue fixed with genipin can provide a natural microenvironment for host cell migration and may be used as a tissue-engineering extracellular matrix (ECM) to accelerate tissue regeneration [6]. Genipin, a naturally occurring crosslinking agent, can be isolated from the fruits of Gardenia jasminoides ELLIS [7]. It is known that the cytotoxicity of genipin is significantly less than that of glutaraldehyde, a commonly used crosslinking agent [8,9]. Genipin has been used to fix biological tissues or amino-group containing biomaterials for biomedical applications [7,10,11]. In the present study, the acellular bovine pericardial tissue was further treated with acetic acid and subsequently with collagenase to increase its pore size and interconnectivity. It is generally accepted that a tissue-engineering ECM must be highly porous for a sufficient cell density to be seeded in vitro, for blood invasion to occur in vivo and for oxygen and nutrients to be supplied to cells [12]. It is known that the human heart cannot regenerate significantly because adult cardiomyocytes are terminally differentiated and cannot replicate after injury [13]. In an attempt to overcome this limitation, the porous acellular bovine pericardium was additionally seeded with mesenchymal stem cells (MSCs) as a patch to repair a surgically created myocardial defect in the right ventricle of a syngeneic rat model. MSCs are bone marrow-derived cells that retain the ability to differentiate into various types of tissue cells and contribute to the regeneration of a variety of mesenchymal tissues including bone, cartilage, muscle and adipose [14,15]. Several studies performed on rodents, pigs as well as on human demonstrated that MSCs own the potency to differentiate into a cardiomyocyte phenotype in the heart [16,17]. 2. Materials and methods 2.1. Experimental animals Animal care and use was performed in compliance with the ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised 1996. Bone marrow MSCs were obtained from syngeneic Lewis inbred rats (LEW/SsNNarl, National Laboratory Animal Center, Taipei, Taiwan) weighing 100–150 g. Adult syngeneic Lewis rats weighing 250–275 g were used for the right ventricular wall replacement.
2.2. Preparation of porous acellular bovine pericardia The procedures used to remove the cellular components from bovine pericardia were based on a method previously reported by Courtman et al. [5] with slight modifications [18]. To increase the pore size and interconnectivity within test samples, the acellular tissues were treated additionally with acetic acid and subsequently with collagenase [18,19]. Afterward, the obtained porous acellular tissues and fresh bovine pericardia before cell extraction were separately fixed in a 0.05% genipin (Challenge Bioproducts, Taichung, Taiwan) aqueous solution (phosphate buffered saline, PBS, pH 7.4) at 37 1C for 3 days. The fixed cellular and
porous acellular tissues were then thoroughly rinsed in PBS. Details of the methodology were previously reported [18,19]. After preparation of test samples, the cellular and porous acellular tissues were processed for light microscopic and scanning electron microscopic (SEM) examinations to investigate their ultrastructures [20]. The pore size of the porous acellular tissues, stained with hematoxylin and eosin (H&E), was determined under a microscope and their porosity was measured by helium pycnometery [21]. The crosslinking degrees of the fixed cellular and porous acellular tissues were determined by measuring their fixation indices and denaturation temperatures (n ¼ 5) [22]. The fixation index, determined by the ninhydrin assay, was defined as the percentage of free amino groups in test tissues reacted with genipin subsequent to fixation. Five tissue strips from each studied group were mechanically examined [22]. To facilitate cell infiltration and repopulation, the dense layer on each side of the porous acellular tissues was sliced off using a cryostat microtome (Leica CM3050S, Leica Microsystems Nussloch GmbH, Nussloch, Germany). The obtained samples were then sterilized in a graded series of ethanol solutions for the following MSC seeding in vitro and the animal study.
2.3. Isolation and culture of bone marrow MSCs Bone marrow MSCs were isolated as described previously [23,24]. Lewis rats were anesthetized with intramuscular administration of ketamine hydrochloride (22 mg/kg) followed by an intraperitoneal injection of sodium pentobarbital (30 mg/kg). Femora and tibia of rats were collected and the adherent soft tissues were carefully removed. Bone marrow was aspirated with a syringe containing 1 mL heparin with a 26-gauge needle and disaggregated into a single-cell suspension by sequential passage through a 26-gauge needle. Mononuclear cells were separated by density-gradient centrifugation over Ficoll-Paque (r ¼ 1:077, Amersham, Uppsala, Sweden) at 1100g for 30 min. The cells were rinsed twice with PBS to remove Ficoll-Paque and then seeded onto plastic tissue culture plates at 1 105 cells/cm2 with culture medium (alpha-Modified Minimum Essential Medium, alpha-MEM, supplemented with 20% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin, GIBCO Laboratory, Life Technologies, Grand Island, NY). Three days later, non-adherent cells were removed by changing the medium. After 14 days in culture, adherent cells formed homogenous fibroblast-like colonies. Adherent cells were passed with 0.05% trypsinEDTA (GIBCO Laboratory) and split at a ratio of 1:3. The culture medium was changed every 3–4 days and the passages 2–4 MSCs were used for the following studies.
2.4. In vitro graft preparation, MSCs seeding onto the porous acellular tissues The obtained MSCs were labeled in vitro for later identification by adding 100 mg/mL 5-bromo-20 -deoxyuridine (BrdU, Sigma-Aldrich Corp., St. Louis, MO) containing media to 50% confluent cultures for 48 h [25]. At the completion of the incubation period, the cells were washed, harvested from culture dishes using 0.05% trypsin solution and resuspended to a concentration of 1 106 bone marrow MSCs in 50 mL of culture medium, and seeded onto the prepared porous acellular tissues (in a disk shape with a diameter of 5 mm and a thickness of 1.6 mm, the MSC patch). Thirty minutes after incubation, drops of culture medium were added at the circumference of the MSC patch. After another 90 min of incubation, 20 mL of culture medium were added to the culture dishes. After incubation for 20 h, the MSC patch was removed from the incubator. To investigate the cell attachment and distribution on the MSC patch, samples were fixed in 10% phosphate-buffered formalin for 2 days and subsequently processed for the H&E stain and SEM examinations.
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2.5. Animal study The prepared MSC patch was used to repair a transmural defect surgically created in the right ventricle of syngeneic rat hearts (n ¼ 10) based on a method reported by Ozawa et al. [26]. The aforementioned genipin-fixed cellular bovine pericardium (sterilized by the same procedure mentioned above) was used as a control (the Control patch, n ¼ 10). Briefly, rats were anesthetized with isoflurane (3.0%) and intubated for continuous ventilation with room air supplement with oxygen and isoflurane (1.5–3.0%). The rat heart was exposed through a median sternotomy. A purse-string suture (5–6 mm in diameter) was placed in the free wall of right ventricle. Each ends of the suture was passed through a 24-gauge plastic vascular cannula (Insyte, Becton Dickinson Vascular Access, Sandy, UT), which was used as a tourniquet. The tourniquet was tightened and the bulging part of the ventricular wall inside the pursestring stitch was lopped off. The tourniquet was transiently loosened to confirm a complete transmural resection. Subsequently, a Control or MSC patch was sutured along the margin of the purse-string stitch with an over-and-over method with 7–0 polypropylene (Prolene, Ethicon, Inc., Somerville, NJ) to cover the defect created in the right ventricle. After completion of suturing, the tourniquet was released and the purse-string stitch was removed. The chest incision was closed in layers with running sutures of 3–0 silk. Finally, rats were removed from ventilation and recovered under a warming lamp.
2.6. Computerized mappings (epicardial electrograms) A 12-lead electrocardiographic system (PC-ECG 1200 M, Norav Medical, Kiryat Bialik, Israel) was used to acquire the epicardial electograms of the implanted patch and its adjacent rat native myocardium immediately after implantation and at retrieval [27].
2.7. Echocardiography (assessment of the cardiac function) Echocardiography was performed before patch implantation and at retrieval for each studied group. Rats were anesthetized with sodium pentobarbital (30 mg/kg) and isoflurane was used as a supplement to maintain mild anesthesia. Cardiac ultrasonography was performed with a commercially available echocardiographic system (SONOS 5500, Agilent Technologies, Andover, MA) equipped with a 12-MHz broadband sector transducer. The heart was imaged in the two-dimensional mode in shortaxis views at the mid-papillary level of left ventricle to evaluate contractions of the Control-patch-implanted or MSC-patch-implanted myocardium at the right ventricle [28]. The implanted samples were retrieved at 4- and 12-week postoperatively (n ¼ 5 per group at each time point) and were used for gross and histological examinations.
2.8. Histological examinations The samples used for light microscopy were fixed in 10% phosphate buffered formalin and prepared for histological examinations. The fixed samples were embedded in paraffin and sectioned into a thickness of 5 mm and then stained with H&E. Also, sections of test samples were stained with Masson’s trichrome or elastic van Gieson (EVG) for the detection of collagen fibrils and muscle fibers or stained with safranin-O to visualize glycolsaminoglycans. Additional sections were stained with a van Gieson solution to visualize mesothelial cells [29]. Immunohistochemical staining with a monoclonal antibody against BrdU (Caltag Laboratories, Burlingame, CA) was used to identify the cells seeded on the MSC patch and revealed by a peroxidase–antiperoxidase technique [30]. Additionally, sections of the retrieved sample were stained with a monoclonal antibody against a-sacromeric actin (clone 5C5, Serotec, Kidlington, Oxford, UK) [31]. A monoclonal antibody against a-smooth muscle actin (a-SMA, DAKO, DAKO Corp., Carpinteria, CA) was used to identify smooth
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muscle cells. Additional sections were stained for factor VIII with immunohistological technique with a monoclonal anti-factor VIII antibody (DAKO) [32]. The density of neo-capillaries in each studied sample was quantified with a computer-based image analysis system (Image-Pros Plus, Media Cybernetics, Silver Spring, MD) and converted to vessels/ mm2 [33]. Five different microscopic fields ( 400 by ECLIPSE-E800, Nikon, Tokyo, Japan) of each patch portion of the right ventricular wall were randomly selected.
2.9. Statistical analysis Statistical analysis for the determination of differences in the measured properties between groups was accomplished using 1-way analysis of variance and determination of CIs, which was performed with a computer statistical program (Statistical Analysis System, Version 6.08, SAS Institute Inc., Cary, NC). All data are presented as mean values7SD.
3. Results 3.1. Preparation of porous acellular bovine pericardia After the genipin fixation, the color of bovine pericardial tissues became bluish. As shown in Fig. 1a, c and e, there was a dense layer on each side of the porous acellular bovine pericardium. After slicing off these dense layers with a cryostat microtome, a porous structure beneath was revealed (Fig. 1b, d and f). The denaturation temperature (74.670.3 1C) and fixation index (59.274.3%) of the fixed bovine pericardium before cell extraction (cellular tissue) were comparable with those (75.370.2 1C and 58.874.8%, respectively, n ¼ 5) after cell extraction. However, the fracture tension value (1.370.3 kN/m) of the porous acellular tissue was significantly lower than that of the cellular tissue (6.670.3 kN/m, n ¼ 5). The pore size and porosity of the porous acellular tissue were 161.8728.3 mm and 95.673.2%, respectively (n ¼ 5). 3.2. Isolation of MSCs and preparation of the MSC patch As shown in Fig. 2a, the isolated MSCs had a fibroblastlike shape, attached to the culture dish tightly, and proliferated in the culture medium. Before seeding onto the porous acellular tissue, the nuclei of MSCs were labeled with BrdU for 48 h, and 88.273.2% (n ¼ 5) of the cultured MSCs were stained positively with BrdU (Fig. 2b). After seeding MSCs onto the porous acellular tissue (the MSC patch, Fig. 2c–f), the cells had uniform, viable fibroblast like morphology (Fig. 2c and e). The MSCs seeded on the patch revealed a good interconnectivity (Fig. 2c). BrdU incorporation was evident in 485% of the seeded MSCs (Fig. 2f). 3.3. Computerized mappings (epicardial electrograms) As shown, immediately after implantation, there were no electrogram signals observed on both the Control and MSC patches (Fig. 3). At 4- and 12-week postoperatively, the mappings of the epicardial electrograms on the Control patch showed abnormalities similar to those observed for
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Fig. 1. Ultrastructures of the anterior [(a) and (b)] and posterior surfaces [(c) and (d)] of porous acellular bovine pericardia, before and after slicing off the dense layer on its each side using a cryostat microtome, together with their cross-sectional views [(e) and (f)] examined by the SEM. The dotted lines in (e) were where the dense layer on each side of the porous acellular bovine pericardium was sliced off.
an old myocardial infarction. In contrast, the electrograms on the epicardial surface of the MSC patch appeared to be normal as compared to those observed on its adjacent native myocardium (Fig. 3).
3.4. Echocardiography (assessment of the cardiac function) Short-axis two-dimensional images of the normal heart (Movie I) and the Control-patch-implanted heart (Movie II) and the MSC-patch-implanted heart (Movie III) at retrieval were obtained using a commercially available echocardiographic system. The results indicated that both the Control (Movie II) and MSC patches (Movie III) were akinetic. It was noted that the differences in global contraction of the native myocardium, the Control-patchimplanted myocardium and the MSC-patch-implanted myocardium at the right ventricle were difficult to differentiate.
3.5. Gross examination No aneurysmal dilation of the implanted patches was seen for both studied groups throughout the entire course of the study. On the outer (epicardial) surface, all studied animals in the Control group at 4-week postoperatively were free of any adhesions (n ¼ 5), while two out of the five studied animals at 12-week postoperatively had a filmy adhesion, attached to part of the patch surface and along the suture line, to the chest wall (Fig. 4a). In contrast, no apparent tissue adhesion was observed for the MSC patch throughout the entire course of the study (Fig. 4b). On the inner (endocardial) surface, intimal thickening was observed for both studied groups; however, no thrombus formation was found (Fig. 4c and d). 3.6. Histological findings At 4-week postoperatively, host cells together with neotissue fibrils and neo-capillaries were clearly observed in the
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Fig. 2. Photomicrographs of bone marrow MSCs in culture (a) and stained with a monoclonal antibody against BrdU [(b), black arrows: BrdU positively stained cells, white arrows: BrdU negatively stained cells] ( 200 magnification); SEM micrograph of the MSC patch (c) and its cross-sectional view (d); Photomicrograph of the MSC patch stained with H&E [ 400 magnification, (e)], and that stained with a monoclonal antibody against BrdU [ 400 magnification, (f)].
inner and outer layers of the MSC patch, an indication of tissue regeneration (Fig. 5b and d). An intact layer of endothelial cells was found on the intimal thickening generated on the inner surface of the MSC patch identified by the factor VIII staining (Fig. 5f). In contrast, endothelial cells did not universally and totally cover the entire inner surface of the Control patch (Fig. 5e). The outer surface of the MSC patch was positively stained with van Gieson (Fig. 5h), indicating the presence of mesothelial cells. However, no mesothelial cells were found on the outer surface of the Control patch (Fig. 5g). Instead, a thick layer of fibrous tissue was firmly attached onto the outer surface of the Control patch (Fig. 5c). In the middle layer of the MSC patch, host cells together with neo-tissue fibrils were found to fill most of its inside pores (Fig. 6b). The neo-tissue fibrils regenerated in the MSC patch were identified to be neo-muscle fibers (stained red) with a few neo-collagen fibrils (stained blue) by the Masson’s trichrome stain (Fig. 6d). The neo-muscle fibers seen in the MSC patch were further confirmed by the EVG
stain (stained brown, Fig. 6f). Also, there were some neoglycosaminoglycans regenerated within the pores of the MSC patch recognized by the safranin-O stain (stained pink, Fig. 6h). In contrast, no apparent tissue regeneration was observed inside the Control patch (Fig. 6a, c, e and g). a-SMA positively stained cells were observed in the MSC patch (Fig. 7a), indicating the presence of smooth muscle cells. Additionally, BrdU-labeled cells were clearly identified (Fig. 7b–d). Some of the identified BrdU-labeled cells were further stained positively for a-sacromeric actin (Fig. 7b), indicating that a portion of the implanted bone marrow MSCs had been differentiated towards the myocytic lineage and expressed cytoplasmic a-sacromeric actin. Moreover, a few capillary walls composed of BrdUlabeled endothelial cells (Fig. 7c) and some BrdU-labeled smooth muscle cells (Fig. 7d) were recognized in the MSC patch. In contrast, no such stained cells were observed in the Control patch. At 12-week postoperatively, there were still no signs of tissue regeneration (no host cells, no capillaries, no
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Fig. 3. Local electrograms obtained from the electrodes located on the epicardial surfaces of the Control and MSC patches and their adjacent rat native myocardia immediately after implantation and those observed at 4 and 12-week postoperatively.
Fig. 4. Photographs of the outer (epicardial) surfaces of the Control and MSC patches [(a) and (b)] and their inner (endocardial) surfaces [(c) and (d)] retrieved at 12-week postoperatively.
neo-tissue fibrils and no cells stained positively for asacromeric actin or a-SMA) observed inside the Control patch. The neo-tissue fibrils seen in the MSC patch appeared to be more compact and organized than their
counterparts observed at 4-week postoperatively. BrdU-, a-sacromeric-actin- and a-SMA- cells were still observed in the MSC patch. The density of neo-capillaries observed in the MSC patch retrieved at 12-week postoperatively
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Fig. 5. Photomicrographs of the inner (endocardial) layers of the Control patch (a) and the MSC patch (b); and the outer (epicardial) layers of the Control patch (c) and the MSC patch (d) stained with H&E retrieved at 4-week postoperatively ( 200 magnification); insets of (b) and (d) magnified an additional 4-fold photomicrographs that stained with H&E, neo-capillaries are indicated by the black arrows. Photomicrographs of the Control and MSC patches stained with an antibody against factor VIII [(e) and (f)]; and van Gieson [(g) and (h)] retrieved at 4-week postoperatively ( 800 magnification).
(335720 vessels/mm2) was comparable to its counterpart found at 4-week postoperatively (327741 vessels/mm2, p40:05); however, it was still much less than that seen in the native myocardium (21187320 vessels/mm2, po0:05). 4. Discussion Cell transplantation is a promising therapy for patients with ischemia and ventricle dysfunction [24,34,35]. However, the engraftment process is limited and a large mature scar does not benefit from cell transplantation alone. Resection of the aneurysm and surgical remodeling of the ventricle to restore chamber size and shape may improve the cardiac function under ideal circumstances [36]. Nevertheless, the benefits of procedures of surgical scar resection and ventricular remodeling may be limited by the use of non-viable synthetic patch materials [36]. At retrieval, it was found that both the Control and MSC patches did not thin and dilate throughout the entire course of the study. This indicated that the mechanical strengths of both studied groups were strong enough to
tolerate the right ventricular pressure of the studied animal and prevented the implanted patches from dilation. At 4-week postoperatively, host cells together with neotissue fibrils and neo-capillaries were clearly observed in the outer (epicardial) layer of the MSC patch, indicating that the outer layer of the MSC patch became well integrated with its host tissues (Fig. 5d). Additionally, an intact layer of neo-mesothelial cells (i.e., completed remesothelialization, identified by the van Gieson stain) was present on top of the neo-tissue fibrils regenerated in the outer layer of the MSC patch (Fig. 5h). However, such observations did not occur for the Control patch (Fig. 5c and g). Instead, a thick layer of fibrous tissue was firmly attached onto the outer surface of the Control patch. It is known that the epicardium forms the outer covering of the heart and has an external layer of flat mesothelial cells [37]. Whitaker et al. [38] reported that a pure culture of mesothelial cells was able to induce fibrinolysis. Another study suggested that the mesothelial fibrinolytic properties are associated with the secretion of tissue plasminogen activator [39]. These results likely explained
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Fig. 6. Photomicrographs of the middle layers of the Control and MSC patches stained with H&E [ 400 magnification, (a) and (b)], Masson’s trichrome [ 400 magnification, (c) and (d)], EVG [ 400 magnification, (e) and (f)], and safranin-O [ 400 magnification, (g) and (h)] retrieved at 4-week postoperatively.
the observation that once the surface of the implanted MSC patch was populated with mesothelial cells, it remained resistant to adhesion formation. An intimal thickening covered with endothelial cells was found on the inner (endocardial) surfaces of the Control and MSC patches (Fig. 5e and f). This finding suggested that host endocardial endothelial cells or endothelial progenitor cells were involved in the endothelialization on the inner surfaces of the implanted patches [4,40]. No thrombus formation was observed on either studied sample. It is known that the most thrombo-resistant graft surface is that provided by normal endothelial cells [41]. The basis for the thrombo-resistance of endothelial cells may possibly be related to their role as a source of plasminogen activator, prostacyclin (PGI2), or both [42]. Plasminogen is a key proenzyme in fibrinolysis and thrombolytic system [43]. In the middle layer of the MSC patch, host cells together with neo-muscle fibers (with a few neo-collagen fibrils), neo-glycosaminoglycans and neo-capillaries were observed inside the pores, indicated by the H&E, Masson’s trichrome, EVG and safranin-O stains (Fig. 6b, d, f
and h), an indication of tissue regeneration. Additionally, a-SMA positively stained cells were seen in the MSC patch, indicating the presence of smooth muscle cells (Fig. 7a). It is known that smooth muscle cells permit formation of a muscular tissue (observed in Fig. 6d and f, stained red by Massson’s trichrome and stained brown by EVG) in addition to collagen formation (stained blue by Massson’s trichrome) [44]. These findings suggested that host progenitor cells from the systemic circulation or the surrounding tissue may be relevant to the presence of smooth muscle cells in the implanted MSC patch [40]. Previous studies reported that smooth-muscle-cell transplantation into myocardial infarct scar tissues improves heart function and prevents ventricle dilatation [34]. On the other hand, some cardiomyocytes, which stained positively for BrdU and a-sacromeric actin, were observed in the MSC patch (Fig. 7b), indicating that the implanted MSCs, transferred within the patch, can engraft and differentiate into cardiomyocytes. It was reported that the MSCs transplanted into a myocardium environment can express myogenic-specific proteins such as a-sacromeric actin, sarcomeric MHC, desmin, troponin T and
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Fig. 7. Photomicrographs of immunohistochemistry staining of a monoclonal antibody against a-smooth muscle actin [ 400 magnification, (a)], a monoclonal antibody against BrdU (stained brown) and a-sacromeric actin (stained blue) [ 2000 magnification, (b)], a monoclonal antibody against BrdU (stained brown) and factor VIII (stained blue) [ 2000 magnification, (c)] and a monoclonal antibody against BrdU (stained brown) and a-smooth muscle actin (stained blue) [ 2000 magnification, (d)] in the MSC patch retrieved at 4-week postoperatively.
phospholamban [24,45]. Our finding confirmed that the adult heart may provide an environment for the cardiomyogenic differentiation of the implanted MSCs. Additionally, it was reported that the MSCs transplanted in the myocardium can be differentiated into endothelial cells and smooth muscle cells [45–47]. This fact was also confirmed in our study. As shown, a few capillary walls composed of BrdU-labeled endothelial cells (Fig. 7c) together with some BrdU-labeled smooth muscle cells (Fig. 7d) were identified in the MSC patch. Since only a few neo-capillaries and smooth muscle cells were positively labeled with BrdU, the majority of the neo-capillaries and smooth muscle cells observed in the MSC patch must be originated from the host progenitor cells in blood or the surrounding tissues. At 12-week postoperatively, the neo-tissue fibrils (neomuscle fibers, neo-glycosaminoglycans and neo-capillaries) observed in the MSC patch were more compact and organized than its counterparts observed at 4-week postoperatively. BrdU-labeled cardiomyocytes, endothelial cells and smooth muscle cells were still observed in the MSC patch. The densities of neo-capillaries found in the MSC patch throughout the entire course of the study were still significantly lower than that observed in the native myocardium. It was reported that neovascularization is important in the long-term survival of the transplanted cells in the scar of heart [24]. The aforementioned results indicated that the MSC patch may preserve the structure of the ventricular wall while providing the potential for growth and contractility. However, the echocardiographic results showed that the
MSC patch was akinetic (Movie III), in spite of the presence of cardiomyocytes and a normality of the local electrograms. This may be attributed to the fact that there were not enough cardiomyocytes present in the MSC patch. Additionally, these cardiomyocytes were not in contact with their host counterparts and therefore were not able to beat synchronously with the host myocardium. The normality of the local electrograms observed on the MSC patch, an indication of a better electrical conductance, may be attributed to the regenerated tissues observed in its inside pores. In contrast, no apparent tissue regeneration was observed in the Control patch (Fig. 6a, c, e and g) throughout the entire course of the study, while only abnormal electrogram signals were observed on its epicardial surface (Fig. 3). It was reported in the literature that one of the major problems of bioprostheses is calcification [48–50]. Although tissue calcification was not observed in both studied patches throughout the entire course of the study, long-term utility of the MSC patch may still need to consider its possible calcification and accompanying mechanical degradation.
5. Conclusions The MSC patch may preserve the structure of the right ventricle and prevent aneurysmal dilation while providing the potential for myocardial tissue regeneration. The MSC patch holds promise to become a suitable patch for surgical repair of damaged myocardium.
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Acknowledgments This work was supported by a grant from Veterans General Hospitals and University System of Taiwan Joint Research Program (VGHUST95-P6-18). Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biomaterials.2006.06.022.
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