International Journal of Cardiology 173 (2014) 410–423

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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

Direct implantation versus platelet-rich fibrin-embedded adipose-derived mesenchymal stem cells in treating rat acute myocardial infarction Cheuk-Kwan Sun a, Yen-Yi Zhen b,1, Steve Leu c, Tzu-Hsien Tsai b, Li-Teh Chang d, Jiunn-Jye Sheu e, Yung-Lung Chen b, Sarah Chua b, Han-Tan Chai b, Hung-I Lu e, Hsueh-Wen Chang f, Fan-Yen Lee e,2, Hon-Kan Yip b,c,⁎ a

Department of Emergency Medicine, E-DA Hospital, I-Shou University, Kaohsiung, Taiwan Division of Cardiology, Department of Internal Medicine, Taiwan c Center for Translational Research in Biomedical Sciences, Taiwan d Basic Science, Nursing Department, Meiho Institute of Technology, Pingtung, Taiwan e Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan f Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan b

a r t i c l e

i n f o

Article history: Received 17 October 2013 Received in revised form 21 January 2014 Accepted 9 March 2014 Available online 14 March 2014 Keywords: Acute myocardial infarction Platelet-rich fibrin scaffold Adipose-derived mesenchymal stem cell Angiogenesis Left ventricular remodeling

a b s t r a c t Background: This study tested whether adipose-derived mesenchymal stem cells (ADMSC) embedded in platelet-rich fibrin (PRF) scaffold is superior to direct ADMSC implantation in improving left ventricular (LV) performance and reducing LV remodeling in a rat acute myocardial infarction (AMI) model of left anterior descending coronary artery (LAD) ligation. Methods: Twenty-eight male adult Sprague Dawley rats equally divided into group 1 [sham control], group 2 (AMI only), group 3 (AMI + direct ADMSC implantation), and group 4 (AMI + PRF-embedded autologous ADMSC) were sacrificed on day 42 after AMI. Results: LV systolic and diastolic dimensions and volumes, and infarct/fibrotic areas were highest in group 2, lowest in group 1 and significantly higher in group 3 than in group 4, whereas LV performance and LV fractional shortening exhibited a reversed pattern (p b 0.005). Protein expressions of inflammation (oxidative stress, IL-1β, MMP-9), apoptosis (mitochondrial Bax, cleaved PARP), fibrosis (Smad3, TGF-β), and pressure-overload biomarkers (BNP, MHC-β) displayed a pattern similar to that of LV dimensions, whereas anti-inflammatory (IL-10), anti-apoptotic (Bcl-2), and anti-fibrotic (Smad1/5, BMP-2) indices showed a pattern similar to that of LV performance among the four groups (all p b 0.05). Angiogenesis biomarkers at protein (CXCR4, SDF-1α, VEGF), cellular (CD31 +, CXCR4+, SDF-1α +), and immunohistochemical (small vessels) levels, and cardiac stem cell markers (C-kit+, Sca-1+) in infarct myocardium were highest in group 4, lowest in group 1, and significantly higher in group 3 than in group 2 (all p b 0.005). Conclusion: PRF-embedded ADMSC is superior to direct ADMSC implantation in preserving LV function and attenuating LV remodeling. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Although the incidence of acute myocardial infarction (AMI) has recently been reported to be steady worldwide with an improved in-hospital mortality [1,2], the short-term and long-term mortality

⁎ Corresponding author at: Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, 123, Dapi Road, Niaosung Dist., Kaohsiung City 83301, Taiwan. Tel.: +886 7 7317123; fax: +886 7 7322402. E-mail address: [email protected] (H.-K. Yip). 1 Indicates equal contribution in this study compared with the first author. 2 Indicates equal contribution in this study compared with the corresponding author.

http://dx.doi.org/10.1016/j.ijcard.2014.03.015 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.

rates remain notably high [3,4], especially in the aged patients [4]. AMI-induced myocardial necrosis and the subsequent progressive left ventricular (LV) remodeling eventually lead to LV chamber dilatation and pump failure that accounts for the poor clinical outcomes [5–7]. Despite the wide acceptance of primary percutaneous coronary intervention and thrombolytic therapy as the gold standards in rescuing the ischemic/jeopardized myocardium [8–10], the loss of myocardium after AMI is always irreversible. Of paramount importance is that the severity of pump failure depends on the extent of myocardial loss and the degree of myocardial ischemia [5–7,11,12]. Maximizing the mass of viable myocardium after infarction through myocardial repair/regeneration of functional myocardium, therefore, is the ultimate therapeutic goal.

C.-K. Sun et al. / International Journal of Cardiology 173 (2014) 410–423

Abundant studies from both experimental models and clinical trials have established the safety and effectiveness of stem cell therapy in improving heart function in the settings of ischemia-related LV dysfunction and myocardial infarction [13–18]. On the other hand, therapeutic success of stem cell treatment against AMI depends on several essential factors, including the type and number of stem cells, time point of stem cell treatment, autologous versus allogenic stem cell utilization, and the route of the stem cell transplantation. Interestingly, the most common method for stem-cell implantation in experimental studies is direct myocardial needle injection of cell suspensions [15,16,19,20]. Albeit simple, this procedure is associated with rapid cell loss from leakage through the injection site, needle-mediated direct tissue damage, or even heart perforation with uncontrollable hemorrhage due to infarctrelated thinning and fibrosis of LV wall. For these reasons, the clinical applicability of direct myocardial injection of cell suspensions has its limitations. Accordingly, the development of a more suitable method for cell administration not only can avoid needle-related complications, but can also ensure more secure contact between the ischemic myocardium and the introduced cells to augment therapeutic outcome. Growing evidence has suggested biological scaffolding with tissue engineering as a potentially applicable strategy for effective delivery of mesenchymal stem cells (MSC) for myocardial repair after myocardial infarction [21,22]. Application of this “patching” technique using autologous grafts embedded with autologous MSCs/endothelial progenitor cells (EPCs) may, therefore, improve LV function and suppress LV remodeling by at least two mechanisms: 1) myocardial regeneration from enhanced angiogenesis, paracrine effects, and possible myocardial differentiation from MSCs/EPCs, and 2) mechanical support from ventriculoplasty to reduce chamber size and, therefore, the wall stress according to the Laplace's law. Moreover, studies have recently shown that adipose tissue is a promising source of autologous stem cells [e.g., adipose-derived mesenchymal stem cell (ADMSC)] in the treatment of ischemia-related organ dysfunction [23,24]. Therefore, using a rodent AMI model, this study attempted to test the following hypotheses: 1) The scaffold made from platelet-rich fibrin (PRF), when patched on the infarct area, might preserve LV function and suppress LV remodeling; 2) Patching of infarcted myocardium with ADMSCs embedded in PRF (i.e., autologous ADMSC graft) may be superior to direct myocardial implantation of ADMSCs in improving LV function and ameliorating LV remodeling.

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40 min of incubation, the content was triturated with a 25 mL pipette for 2–3 min. The cells obtained were placed back to the rocker for incubation. The contents of the flask were transferred to 50 mL tubes after digestion, followed by centrifugation at 600 ×g, for 5 min at room temperature. The fat layer and saline supernatant from the tube were poured out gently in one smooth motion or removed using vacuum suction. The cell pellet thus obtained was resuspended in 40 mL saline and then centrifuged again at 600 ×g for 5 min at room temperature. After being resuspended again in 5 mL saline, the cell suspension was filtered through a 100 μm filter into a 50 mL conical tube to which 2 mL of saline was added to rinse the remaining cells through the filter. The flow-through was pipetted to a 40 μm filter into a new 50 mL conical tube. The tubes were centrifuged for a third time at 600 ×g for 5 min at room temperature. The cells were resuspended in saline. Isolated ADMSCs were cultured in a 100 mm diameter dish with 10 mL DMEM culture medium containing 10% FBS for 14 days. 2.3. Preparation of PRF, ADMSC labeling, AMI induction, and patching By day 14 after ADMSC isolation and culture, CM-Dil (Vybrant™ Dil cell-labeling solution, Molecular Probes, Inc.) (50 μg/mL) was added to the culture medium 30 min before AMI induction for ADMSC (1.0 × 106 cells per batch) labeling as previously reported [24,25]. At the same time, 3.0 mL of blood was drawn through cardiac puncture from rat and placed in an Eppendorff tube, followed by centrifugation (model 5415D; Eppendorf) at 400 g for 10 min at room temperature. The suspension of white jelly-like component, so-called “PRF scaffold”, was collected after centrifugation (Fig. 1A, B, & C). The scaffold was then cut into two pieces each of which was placed at the bottom of an Eppendorff tube. Two batches of ADMSCs (0.5 × 106 each) previously labeled with CM-DiI were pipetted onto the two pieces of PRF in the Eppendorff tubes, respectively, before being centrifuged at 800 g for 20 min. This procedure helped in the embedment of ADMSCs into the PRF scaffold. During the preparation of PRF scaffold and ADMSC grafts, all animals were anesthetized by inhalational 2.0% isoflurane and placed in a supine position on a warming pad at 37 °C. Under sterile conditions, the heart was exposed via a left thoracotomy. AMI was induced in group 2 to 4 animals by left coronary artery ligation 3 mm distal to the margin of left atrium with a 7–0 prolene suture. Regional myocardial ischemia was verified by observing a rapid color change from pink to dull red over the anterior surface of the LV and rapid development of akinesia and dilatation over the affected region. Rats receiving thoracotomy only without AMI induction served as sham controls (group 1). For group 4 animals, ADMSCs were embedded into the PRF scaffolds through centrifugation of the scaffolds in an Eppendorf tube with ADMSCs placed on top of the graft so that the cells sank into the graft through the gravitational force of centrifugation. Care was then taken to place the upper side of the graft (i.e. the side with direct contact with ADMSCs during centrifugation) on the heart before securing the graft in place with interrupted 7/0 prolene sutures over the edge of the graft so that the embedded cells were in direct contact with the heart. Two pieces of ADMSC-embedded grafts were used to ensure maximum cell embedding during centrifugation and also to completely cover the infarct surface of LV myocardium to avoid undue tension of using a single graft that may restrict cardiac dilatation (Fig. 1D, E, & F). For group 3 animals, ADMSCs were directly implanted into the infarct region at four different points with two over the upper and two over the lower zones. After the procedure, the thoracotomy wound was closed and the animals were allowed to recover from anesthesia in a portable animal intensive care unit (ThermoCare®) for 24 h.

2. Materials and methods 2.4. Functional assessment by echocardiography 2.1. Ethics All animal experimental procedures were approved by the Institute of Animal Care and Use Committee at Chang Gung Memorial Hospital-Kaohsiung Medical Center (Affidavit of Approval of Animal Use Protocol No. 2010122405) and performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23, National Academy Press, Washington, DC, USA, revised 1996). 2.2. Animal grouping and isolation of adipose tissue for culturing adipose-derived mesenchymal stem cells Pathogen-free, adult male Sprague–Dawley (SD) rats (n = 28) weighing 325–350 g (Charles River Technology, BioLASCO Taiwan Co. Ltd., Taiwan) were randomized and equally categorized into group 1 (sham controls receiving thoracotomy only, n = 7), group 2 (AMI induction only, through left coronary artery ligation, n = 7), group 3 (AMI + autologous ADMSC, n = 7), and group 4 (AMI + autologous ADMSCembedded PRF scaffold, n = 7). The dosage of ADMSC (2.0 × 106) was chosen according to our previous studies with minor modifications [15,20,24]. Rats in groups 3 and 4 were anesthetized with inhalational 2% isoflurane 14 days before AMI induction for harvesting peri-epididymal adipose tissue. The procedure and protocol for the culture and identification of ADMSCs were described in our recent reports [24,25]. Briefly, the adipose tissue surrounding the epididymis was carefully dissected and excised. Then 200–300 μL of sterile saline was added to every 0.5 g of tissue to prevent dehydration. The tissue was cut into b1 mm3 pieces using a pair of sharp, sterile surgical scissors. Sterile saline (37 °C) was added to the homogenized adipose tissue in a ratio of 3:1 (saline: adipose tissue), followed by the addition of stock collagenase solution to a final concentration of 0.5 units/mL. The tubes with the contents were placed and secured on an orbital shaker and incubated with constant agitation for 60 ± 15 min at 37 °C. After

All animals underwent transthoracic echocardiography under general anesthesia in supine position at the beginning and end of the study. The procedure was performed by an animal cardiologist blind to the experimental design using an ultrasound machine (Vevo 2100, Visualsonics). M-mode standard two-dimensional (2D) left parasternallong axis echocardiographic examination was conducted. Left ventricular internal dimensions [end-systolic diameter (ESD) and end-diastolic diameter (EDD)] were measured at mitral valve and papillary levels of left ventricle, according to the American Society of Echocardiography leading-edge method using at least three consecutive cardiac cycles. LVEF was calculated as follows: LVEF (%) = [(LVEDD3 − LVEDS3) / LVEDD3] × 100%. 2.5. Western blot analysis of heart tissue The procedure and protocol for Western blot analysis were based on our recent reports [20,23,24]. Briefly, equal amounts (50 μg) of protein extracts were loaded and separated by SDS-PAGE using acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific sites were blocked by incubation of the membrane in blocking buffer [5% nonfat dry milk in T-TBS (TBS containing 0.05% Tween 20)] overnight. The membranes were incubated with the indicated primary antibodies [Bax (1:1000, Abcam), cleaved poly (ADP-ribose) polymerase (PARP) (1:1000, Cell Signaling), Bcl-2 (1:200, Abcam), phosphorylated (p)-Smad3 (1:1000, Cell Signaling), p-Smad1/5 (1:1000, Cell Signaling), bone morphogenetic protein (BMP) 2 (1:5000, Abcam), transforming growth factor (TGF)-β (1:500, Abcam), interleukin (IL)-1β (1:1000, Cell Signaling), matrix metalloproteinase (MMP)-9 (1:3000, Abcam), IL-10 (1:5000, Abcam), CXCR4 (1:1000, Abcam), stromal cell-derived factor (SDF)-1α (1:1000, Cell Signaling), endothelial nitric oxide synthase (eNOS) (1:1000, Abcam), vascular endothelial cell growth factor (VEGF) (1:1000, Abcam), brain natriuretic peptide (BNP) (1:600, Abcam), myosin

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Fig. 1. Preparation of platelet-rich fibrin (PRF) scaffold and PRF-embedded adipose-derived mesenchymal stem cells (ADMSCs) and patching of PRF on left ventricular myocardial surface. A & B) Preparation of platelet-rich fibrin (PRF) scaffold, white jelly-like component (outlined by yellow dotted line) by centrifugation. C) Example of a PRF graft with embedded ADMSCs ready for patching. D & E) Schematic illustration of peri-infarct area, infarct area, and two PRF grafts with ADMSCs covering the left ventricular (LV) myocardial surface secured in place by sutures.

heavy chain (MHC)-α (1:300, Santa Cruz), MHC-β (1:1000, Santa Cruz), and actin (1:10000, Chemicon)] for 1 h at room temperature. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1:2000, Cell Signaling) was used as a secondary antibody for 1-h incubation at room temperature. The washing procedure was repeated eight times within 1 h. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) and exposed to Biomax L film (Kodak). For purposes of quantification, ECL signals were digitized using Labwork software (UVP). 2.6. Oxidative stress reaction in LV myocardium The procedure and protocol for assessing the protein expression of oxidative stress have previously been described in details in our previous reports [20,23,24]. The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). DNPH derivatization was carried out on 6 μg of protein for 15 min according to the manufacturer's instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1: 150) for 2 h, followed by incubation in secondary antibody solution (1:300) for 1 h at room temperature. The washing procedure was repeated eight times within 40 min. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP). For oxyblot protein analysis, a standard control was loaded on each gel. 2.7. Immunofluorescent (IF) and immunohistochemical (IHC) staining IF staining was performed for the examinations of CD31+, CXCR4+, and SDF-1α+ cells as well as troponin-I in LV myocardium using respective primary antibodies based

on our recent study [25]. Moreover, IHC staining was performed for examinations of c-Kit and Sca-1 using respective primary antibodies as described [25]. Irrelevant antibodies were used as controls in the current study. 2.8. Vessel density and arterial muscularization in LV infarct area IHC staining of small blood vessels was performed with α-SMA (1:400) as primary antibody at room temperature for 1 h, followed by washing with PBS thrice. Ten minutes after the addition of anti-mouse-HRP conjugated secondary antibody, the tissue sections were washed with PBS thrice. Then 3,3′ diaminobenzidine (DAB) (0.7 g/tablet) (Sigma) was added, followed by washing with PBS thrice after one minute. Finally, hematoxylin was added as a counter-stain for nuclei, followed by washing twice with PBS after 1 min. Three heart sections were analyzed in each rat. For quantification, three randomly selected high power fields (HPFs) (200×) were analyzed in each section. The mean number per HPF for each animal was then determined by summation of all numbers divided by 9. Muscularization of the arterial medial layer (i.e., an index of vascular remodeling) in LV infarct area was defined as a mean thickness of vessel wall greater than 50% of the luminal diameter in a vessel of diameter N 50 μm. Measurement of arteriolar diameter and wall thickness was achieved using the Image-J software (NIH, Maryland, USA). 2.9. Histological quantification of myocardial fibrosis/infarct Masson's trichrome staining was used for studying fibrosis of LV myocardium. Three serial sections of LV myocardium were prepared at 4 μm thickness by Cryostat (Leica CM3050S). The integrated area (μm2) of fibrosis in the slides was calculated using Image Tool 3 (IT3) image analysis software (University of Texas, Health Science Center, San Antonio, UTHSCSA; Image Tool for Windows, Version 3.0, USA). Three selected sections were quantified for each animal. Three randomly selected high-power fields (HPFs)

C.-K. Sun et al. / International Journal of Cardiology 173 (2014) 410–423 (100×) were analyzed in each section. After determining the number of pixels in each fibrotic area per HPF, the numbers of pixels obtained from the three HPFs were summed. The procedure was repeated in two other slides for each animal. The mean pixel number per HPF for each animal was then determined by summating all pixel numbers and dividing by 9. The mean integrated area (μm2) of fibrosis in LV myocardium per HPF was obtained using a conversion factor of 19.24 (1 μm2 represented 19.24 pixels). The method of calculating the integrated area (μm2) of infarct area in LV myocardium in the tissue sections was identical to that for the integrated area (μm2) of fibrotic area using Image Tool 3 (IT3) image analysis software.

2.10. Statistical analysis Quantitative data are expressed as means ± SD. Statistical analysis was adequately performed by ANOVA followed by Bonferroni multiple-comparison post hoc test. SAS statistical software for Windows version 8.2 (SAS institute, Cary, NC) was utilized. A probability value b0.05 was considered statistically significant.

3. Results 3.1. Echocardiography findings prior to and after AMI induction and anatomical findings of cross-section infarct area Table 1 shows the transthoracic echocardiographic findings of the four groups of animals. Prior to AMI induction, all echocardiography parameters, including thickness of interventricular septum (IVS) and posterior wall, LV end-diastolic dimension (LVEDd), LV end-systolic dimension (LVESd), LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), LV fractional shortening (LVFS) and LV ejection fraction (LVEF) did not differ among the four groups. Additionally, at the end of study period (i.e., by day 42 after AMI induction), the thickness of posterior wall was also similar among the four groups. However, IVS was thinnest in group 2 and thickest in group 1, and significantly thinner in group 3 and group 4 than that in group 1. On the other hand, LVESd was largest in group 2 and smallest in group 1, and significantly larger in group 3 and group 4 than that in group 1. Nevertheless, no significant difference in these two parameters was noted between group 3 and group 4. In addition, LVEDd, LVEDV, and LVESV were laargest in group 2, smallest in group 1, and significantly larger in group 3 than that in group 4. On the other hand, LVFS and LVEF showed an opposite pattern compared to that of LVEDd, LVEDV, and LVESV among the four groups. Moreover, the cross-sectional myocardial infarct area at papillary muscle level was largest in group 2, significantly larger in groups 3 and 4 than that in group 1 (i.e., non-infarct group), and significantly larger in group 3 than that in group 4.

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3.2. Histopathological findings of LV infarct area Masson's Trichrome staining revealed that the mean fibrotic area was largest in group 2, smallest in group 1, and significantly larger in group 3 than that in group 4 (Fig. 2A–E). Moreover, hematoxylin and eosin (H & E) staining showed that the pattern of variation in mean infarct area (IA) was identical to that of fibrotic area among the four groups (Fig. 2F–J). Furthermore, the number of muscularized arterioles was notably higher in groups 2 and 3 than that in groups 1 and 4, but it showed no difference between groups 2 and 3 or between groups 1 and 4 (Fig. 2K). 3.3. IHC and IF studies of angiogenesis biomarkers in LV infarct area (Figs. 3, 4 & 5) IF staining showed that the number of cells positive for CD31, an indicator of endothelial cell, was highest in group 4 and lowest in group 2, and significantly higher in group 3 than that in group 1 (Fig. 3A–E). Besides, IHC staining of smooth muscle actin demonstrated that the differences in number of small vessels (b 15 μm in diameter) in IA, an index of angiogenesis/neovascularization, expressed an identical pattern compared to that of CD31-positive cells among the four groups (Fig. 3F–J). IHC staining revealed that the numbers of Sca-1+ and c-Kit+ cells, two cardiac stem cell markers in the IA, were highest in group 4 and lowest in group 1, and significantly higher in group 3 than that in group 2 (Fig. 4). Furthermore, IF staining showed that the difference in the number of cells positive for CXCR4 and SDF-1α, two markers of endothelial progenitor cells (EPCs), was identical to the pattern of change in the number of Sca-1+ and C-kit+ cells among the four groups (Fig. 5). Of importance is that DiI-positive ADMSCs and the cells presenting with positive double staining (i.e., DiI + together with CD31, CXCR4, or SDF-1α) were found to be present not only in PRF scaffold but also in LV myocardium. 3.4. Protein expressions of inflammatory and anti-inflammatory biomarkers in LV infarct area The protein expressions of oxidized protein (Fig. 6A), MMP-9 (Fig. 6B), IL-1β (Fig. 6C), three inflammatory biomarkers, were highest in group 2 and lowest in group 1, and significantly higher in group 3 than that in group 4. On the other hand, the protein expression of IL-10 (Fig. 6D), an anti-inflammatory index, was highest in group 4

Table 1 Transthoracic echocardiography findings of prior to and after AMI induction among four groups of animals. Variables

Group 1 (n = 7)

Group 2 (n = 7)

Group 3 (n = 7)

Group 4 (n = 7)

Pre-AMI induction Inter-ventricular septum (cm) Posterior wall (cm) LVE-diastolic dimension (cm) LVE-systolic dimension (cm) LVE-diastolic volume (cm) LVE-systolic volume (cm) LV fractional shortening (%) LV ejection fraction (%)

1.54 1.58 8.07 4.79 350.9 106.1 40.82 69.76

± ± ± ± ± ± ± ±

0.18 0.22 0.83 0.76 61.2 41.2 5.23 4.83

1.52 1.68 7.96 4.77 340.3 106.1 40.01 68.82

± ± ± ± ± ± ± ±

0.29 0.21 0.56 0.93 75.1 38.4 4.93 3.21

1.49 1.51 7.88 4.84 335.7 108.9 38.89 67.48

± ± ± ± ± ± ± ±

0.25 0.32 0.48 0.37 87.4 32.6 5.23 3.42

1.58 1.57 7.84 4.52 329.9 95.1 41.01 71.19

± ± ± ± ± ± ± ±

0.23 0.23 0.72 0.64 74.1 45.1 6.43 7.24

At 6 weeks after AMI induction Inter-ventricular septum (cm) Posterior wall (cm) LVE-diastolic dimension (cm) LVE-systolic dimension (cm) LVE-diastolic volume (cm) LVE-systolic volume (cm) LV fractional shortening (%) LV ejection fraction (%)

1.55 1.54 8.23 4.83 367.4 108.9 41.33 70.36

± ± ± ± ± ± ± ±

0.17a 0.2 0.51a 0.42a 71.4a 38.4a 2.64a 3.41a

0.88 1.27 9.39 7.19 490.6 271.4 23.39 44.7

± ± ± ± ± ± ± ±

0.66b 0.32 0.43b 0.41b 57.1b 34.2b 2.71b 4.51b

1.20 1.36 9.06 6.69 453.4 230.4 26.06 50.1

± ± ± ± ± ± ± ±

0.11c 0.12 0.67c 0.52b 82.4c 43.5c 2.47c 5.23c

1.21 1.48 8.67 6.03 411.9 182.4 30.38 55.72

± ± ± ± ± ± ± ±

0.17c 0.52 0.43d 0.46c 63.4d 41.3d 2.97d 4.51d

p-Value 0.911 0.773 0.726 0.783 0.461 0.264 0.579 0.691 b0.001 0.234 0.004 b0.001 0.003 b0.001 b0.001 b0.001

Group 1 = sham control; Group 2 = acute myocardial infarction (AMI) only; Group 3 = AMI + adipose-derived mesenchymal stem cells (ADMSC); Group 4 = AMI + ADMSC + plateletrich fibrin (PRF). LVE = left ventricular end. Letters (a, b, c, d) indicate significant difference (at 0.05 level) by Bonferroni multiple-comparison post hoc test.

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Fig. 2. Left ventricular myocardial histopathological changes on post-infarct day 42 (n = 7). A to D) Microscopic (100×) identification of myocardial fibrosis over left ventricle (LV) after Masson's Trichrome staining. E) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. Scale bars in right lower corners represent 100 μm. F to I) H & E staining for investigation of infarct area and number of muscularized vessels in infarct area of left ventricular (LV) myocardium in four groups of animals. Identification of arterioles under 100× magnification (i.e., square outlined by dotted lines) and examination of arteriolar histology under 600× magnification (i.e., square outlined by solid lines) in the four groups of animals. J) Microscopic (100×) quantification of infarct area: * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. K) Number of muscularized vessels: * vs. other groups with different symbols (*, †, ‡), p b 0.001. Scale bars in right lower corners represent 100 μm. Statistical analysis using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). HPF = high-power field; SC = sham control; AMI = acute myocardial infarction; ADMSC = adipose-derived mesenchymal stem cell; PRF = platelet-rich fibrin.

and lowest in group 1, and significantly higher in group 3 than that in group 2. 3.5. Protein expressions of fibrotic, anti-fibrotic, apoptotic and antiapoptotic biomarkers in LV infarct area The protein expressions of TGF-β and Smad3, two indicators of myocardial fibrosis, were highest in group 2 and lowest in group 1, and significantly higher in group 3 than that in group 4 (Fig. 7A & B). Consistently, the protein expressions of Smad1/5 and BMP-2, two biomarkers of anti-fibrotic activities, exhibited a reversed pattern compared to that of the fibrotic biomarkers among the four groups (Fig. 7C & D). Moreover, the protein expressions of mitochondrial Bax and cleaved (i.e., active form) PARP, two indices of apoptosis, displayed an identical pattern compared to that of fibrotic biomarkers (Fig. 7E & F). On the other hand, the protein expression of Bcl-2, an anti-apoptotic biomarker, showed an opposite pattern compared to that of fibrotic biomarkers among the four groups (Fig. 7G). 3.6. Protein expressions of heart failure and biomarkers of hypertrophic cardiomyopathy in LV infarct area The protein expression of BNP, an indicator of heart failure (i.e., due to pressure or volume overload), was highest in group 2 and lowest in

group 1, and significantly higher in group 3 than that in group 4 (Fig. 8A). Moreover, since cardiac hypertrophy is characterized by fetal β-MHC gene program re-activation, a switch of gene expression from α-myosin heavy chain (α-MHC) to β-MHC can serve as a molecular indicator of the severity of cardiac hypertrophy [26,27]. The expression of β-MHC was highest in group 2 and lowest in group 1, and significantly higher in group 3 than that in group 4 (Fig. 8B). Conversely, the protein expression of α-MHC showed an opposite pattern compared to that of β-MHC among the four groups (Fig. 8C). 3.7. Protein expressions of angiogenic factors in LV infarct area The protein expressions of CXCR4, SDF-1α, and VEGF, three biomarkers of angiogenesis, were lowest in group 1 and highest in group 4, and significantly higher in group 3 than those in group 2 (Fig. 9A–C). In addition, the protein expression of eNOS, an index of endothelial integrity, was highest in group 1 and lowest in group 2, and significantly higher in group 4 than that in group 3 (Fig. 9D). 3.8. Expressions of inflammatory cells in LV infarct area To elucidate the immunogenicity of fibrin-embedded ADMSC, IHC and IF stains were performed. The results demonstrated that the infiltrations of CD3 + (Fig. 10A–E), CD40 + (Fig. 10F–J), CD68 +

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Fig. 3. Microscopic findings of immunofluorescent (IF) and immunohistochemical (IHC) staining for endothelial cells and number of small vessels on post-infarct day 42 (n = 7). A to D) IF microscopic (200×) identification of CD31+ cells (white arrows) in infarct area (IA) of left ventricular (LV) myocardium. Red color (C, D) indicated direct implantation of Dil-stained adipose-derived mesenchymal stem cells (ADMSCs) (C) into LV myocardium or their migration into LV myocardium (D) from epicardial platelet-rich fibrin (PRF) with previously embedded ADMSCs. Merged image (C, D) from double staining (Dil + CD31) demonstrating cellular elements with mixed red-green color (yellow arrows) under high magnifications (600×) (i.e., solid-line squares magnified from small squares with dotted outlines), indicating differentiation of some ADMSCs into endothelial cells (CD31+). Scale bars in right lower corners represent 50 μm. E) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. F to I) IHC staining (α-smooth muscle actin staining) in IA of LV myocardium for identification of small vessels (b15 μm) (black arrows) microscopically (200×) in the four groups of animals. Scale bars in right lower corners represent 50 μm. J) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. HPF = high-power field. Statistical analysis for (E) and (J) using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; ADMSC = adipose-derived mesenchymal stem cell; PRF = platelet-rich fibrin.

(Fig. 10K–O) and CD19+ (Fig. 10P–T) cells, four indices of inflammatory cells, were highest in group 2 and lowest in group 1, and significantly higher in group 3 than that in group 4.

results of this study highlight the potential clinical application of ADMSC-embedded PRF scaffold in the management of ischemic heart disease.

4. Discussion

4.1. Therapeutic use of ADMSC in improving LV function and suppressing LV remodeling

This study, which investigated whether the new technique of ADMSC-embedded PRF scaffold patching was superior to the traditional method of direct ADMSC implantation in (1) preserving LV function, and (2) inhibiting LV remodeling in a rat AMI model, yielded several striking pre-clinical implications. First, as compared with the case of untreated AMI, both therapeutic strategies effectively achieved the two purposes in the present study. Second, the results of the current study suggest that the therapeutic benefits of the two approaches could probably be attributed to the mechanisms of angiogenesis/ neovascularization, anti-inflammation, anti-apoptosis, and anti-fibrosis. Third, of distinct importance is that ADMSC-embedded PRF scaffold application was superior to direct ADMSC implantation in the improvement of LV function and reduction of LV remodeling. Therefore, the

An important finding in the present study is that, as compared with untreated AMI, traditional method of direct ADMSC implantation into infarct myocardium significantly augmented LVEF and LVFS. On the other hand, the parameters of LVEDd, LVESd, LVEDV and LVESV were substantially reduced after treatment. These findings support those of previous experimental studies and clinical trials that stem cell therapy not only improves ischemia-related heart dysfunction, but also effectively inhibits LV remodeling, thereby improving prognostic outcomes [13–20]. The most important finding in the present study is that, as compared with the traditional method of direct ADMSC implantation, ADMSC-embedded PRF scaffold offered an additional benefit of preserving LV thickness and function as well as ameliorating LV remodeling.

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Fig. 4. Immunohistochemical (IHC) staining for identification of myocardial mesenchymal stem cells in infarct area of left ventricular (LV) myocardium on post-infarct day 42 (n = 7). A to D) Microscopic (200×) identification of Sca-1+ cells (black arrows) in LV myocardium after IHC staining. E) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. F to I) Microscopic (200×) identification of c-Kit+ cells (black arrows) in LV myocardium after IHC staining. J) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. HPF = high-power field. Statistical analysis for (E) and (J) using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; ADMSC = adipose-derived mesenchymal stem cell; PRF = platelet-rich fibrin.

Our findings may, therefore, support the clinical use of ADMSCembedded PRF as a therapeutic alternative to direct ADMSC administration in the setting of myocardial infarction.

inhibiting inflammatory reaction, oxidative stress, and LV remodeling as well as in preserving heart function also extend the findings of previous studies [10–20,28–32].

4.2. Anti-inflammatory role of ADMSC treatment in improving LV function and suppressing LV remodeling

4.3. Role of angiogenesis and neovascularization in ADMSC treatment against myocardial infarction

Previous studies have demonstrated that AMI initiates vigorous inflammatory reaction through complement activation and reactive oxygen species (ROS) generation that perpetuate further myocardial damage [28–30]. Accordingly, one important finding of the present study is that the protein (IL-1β, MMP-9) and cellular (CD3+, CD40+, CD68+, CD19 +) expressions of inflammatory, and oxidative stress (oxidized protein) biomarkers in IA were remarkably enhanced, whereas the protein expression of anti-inflammatory biomarker (IL-10) was notably reduced in animals with untreated AMI compared to the sham controls. Therefore, our findings strengthened those of the previous studies [28–30]. In addition, the remarkable reduction in the expressions of inflammatory parameters and enhancement in the expression of antiinflammatory biomarkers following direct ADMSC implantation in this study corroborate the previously reported anti-inflammatory and immunomodulatory properties of ADMSCs [31,32]. Our findings, therefore, in addition to supporting those of previous studies [28–32], also partially explain the improvement in LV function and remodeling after direct ADMSC implantation. The distinct finding of the superiority of using ADMSC-embedded PRF scaffold over direct ADMSC implantation in

Stem cell therapy, which promotes angiogenesis/neovascularization, has been demonstrated to play an essential role in improving ischemiarelated organ dysfunction [15,16,20,23,25]. Consistently, the present study also showed that direct ADMSC implantation enhanced the expressions of angiogenesis factors at protein (CXCR4, SDF-1α and VEGF) and cellular (CXCR4 +, SDF-1α +, and CD31 + cells) levels in IA. Additionally, α-SMA staining revealed that, as compared with untreated AMI, direct ADMSC implantation significantly increased the number of small vessels in IA. Moreover, the protein expression of eNOS, an indicator of the integrity of endothelial cell function, was significantly down-regulated in animals with AMI without treatment compared to that in the sham controls and was up-regulated in AMI animals after receiving direct ADMSC implantation. Therefore, our results strengthened the findings of previous studies [15,16,20,23,25]. Of particular importance is the finding that ADMSC-embedded PRF scaffold further enhanced angiogenesis as reflected in the increased number of small vessels and enhanced integrity of endothelial cell function. Furthermore, results of immunohistochemical staining in the present study highlights the possibility that some PRF-embedded ADMSCs

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Fig. 5. Immunofluorescent (IF) staining for identification of endothelial progenitor cells in infarct area of left ventricular (LV) myocardium on post-infarct day 42 (n = 7). A to D) IF microscopic (400×) identification of SDF-1α+ cells (squared areas). Red color in (C, D) indicated direct implantation of Dil-stained ADMSCs into LV myocardium (C) or their migration into LV myocardium from epicardial platelet-rich fibrin (PRF) with embedded ADMSCs. Merged image (C, D) from double staining (Dil + SDF-1α) demonstrating cellular elements with mixed red-green color under high magnifications (600×) (i.e., solid-line squares magnified from small squares with dotted outlines), indicating differentiation of some ADMSCs into endothelial progenitor cells (EPCs) (SDF-1α+). Scale bars in right lower corners represent 20 μm. E) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. F to I) IF microscopic (400×) identification of CXCR4+ cells in LV myocardium. Faint yellowish color (H) and red color in (I) indicating direct implantation of the Dil-stained ADMSCs into myocardium (H) or migration into LV myocardium (I) from epicardial PRF. Merged image (H, I) from double staining (Dil + SDF-1α) demonstrating cellular components with mixed red-green color (yellow arrows) under high magnifications (600×) (i.e., solid-line squares magnified from small squares with dotted outlines), indicating differentiation of some ADMSCs into EPCs (SDF-1α+). J) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. HPF = high-power field. Statistical analysis for (E) and (J) using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; ADMSC = adipose-derived mesenchymal stem cell; PRF = platelet-rich fibrin.

actually migrated into the myocardium from the epicardium and some of them already differentiated into EPCs (Fig. 3). These findings could, at least in part, explain the further preservation in heart function in animals after ADMSC-embedded PRF scaffold treatment compared to those receiving direct ADMSC implantation. 4.4. Role of anti-apoptosis and anti-fibrosis in ADMSC therapy against myocardial infarction It is well recognized that cellular apoptosis and myocardial fibrosis always occur after AMI [15,16,20]. The resulting progressive cardiomyocyte hypertrophy (i.e., micro-structural change) and LV chamber dilatation (i.e., macro-structural alternation) followed by LV dysfunction and ultimately pump failure associated with elevation of circulating heart failure biomarkers such as BNP are frequently observed in the setting of AMI [5–7]. A principal finding in the present study is that the expressions of apoptotic (mitochondrial Bax, cleaved PARP) and fibrotic (Smad3, TGF-β) biomarkers were substantially increased, whereas those of the anti-apoptotic (Bcl-2) and anti-fibrotic (Smad1/5, BMP-2) biomarkers were notably reduced in AMI animals without treatment

compared with those in the sham controls. In addition, histopathological findings of IA (by H & E staining) revealed substantially increased fibrotic area (by Masson's trichrome staining) in AMI animals without treatment compared to that in the sham controls. However, the patterns of expressions of these parameters were significantly reversed in AMI animals with direct ADMSC implantation. The changes were even more marked in AMI animals receiving ADMSC grafts compared with those without treatment. These findings could, once again, explain the further reduction in LV remodeling as well as preservation in LV wall thickness and function in animals treated with ADMSC-embedded PRF scaffold compared with those receiving direct ADMSC implantation. 4.5. Impact of ADMSC treatment on LV remodeling — expressions of BNP, α-MHC and β-MHC Up-regulation of β-MHC gene expression and down-regulation of α-MHC gene expression have been reported to be specific indicators of cardiac hypertrophy [26,27]. Besides, circulating BNP level has been shown to be a specific biomarker of heart failure in various clinical settings and also an important parameter for predicting prognostic

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Fig. 6. Protein expressions of oxidative stress, inflammation, and anti-inflammation in infarct area of left ventricular (LV) myocardium on post-infarct day 42 (n = 7). A) Oxidative index (protein carbonyls) among the four groups of animals (1 = sham control, 2 = AMI, 3 = AMI + ADMSC, 4 = AMI + PRF + ADMSC). Statistical analysis: * vs. other groups with different symbols (*, †, ‡, §), p b 0.001. (MW: Molecular weight marker; DNP: 1–3 dinitrophenylhydrazone control oxidized molecular protein standard.) B) Protein expression of matrix metalloproteinase (MMP)-9. * vs. other groups with different symbols (*, †, ‡, §), p b 0.001. C) Protein expression of interleukin (IL)-1β. * vs. other groups with different symbols (*, †, ‡, §), p b 0.005. D) Protein expression of IL-10. * vs. other groups with different symbols (*, †, ‡, §), p b 0.001. Statistical analysis using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; ADMSC = adipose-derived mesenchymal stem cell; PRF = platelet-rich fibrin.

outcome after heart failure [33,34]. Intriguingly, the findings from the present study revealed that the protein expressions of BNP and β-MHC were notably increased in AMI animals without treatment compared with those in the sham controls. The expressions of these two parameters were remarkably attenuated in AMI animals with direct ADMSC implantation and further suppressed in AMI animals after receiving ADMSC grafts. On the contrary, the protein expression of α-MHC exhibited an opposite pattern compared to that of β-MHC among all groups of animals. Our findings, in addition to corroborating those of previous studies [27,33,34], once more support a better therapeutic potential of ADMSC grafts than that of direct ADMSC implantation in reducing LV remodeling in a rodent model of AMI at cellular and molecular levels. 4.6. Superiority of ADMSC-embedded prf scaffold patching over direct ADMSC implantation for improving LV function and inhibiting LV remodeling — the reasonable explanations Interestingly, experimental studies have recently shown that using stem cells growing on a sheet as a confluent monolayer to patch on the surface of infarcted LV myocardium significantly improved heart

function [35,36]. The unique finding in the current study is that the use of ADMSC-embedded PRF scaffold was superior to direct ADMSC implantation in preserving heart function and inhibiting the productions of inflammation, apoptosis, fibrosis, oxidative stress, and LV remodeling after AMI. Accordingly, our results strengthen those of previous studies [35,36]. Although we remain uncertain regarding the exact underlying mechanisms involved, the following explanations are considered reasonable. First, as compared to direct implantation of ADMSC into the ischemic myocardium, PRF scaffold could provide a more suitable environment for relatively long ADMSC survival. Second, based on the Laplace's law that the wall stress is directly proportional to the fourth power of the chamber radius, patching of PRF scaffold on the infarct LV surface may provide mechanical support (Supplementary data S1) that helps in limiting LV dilatation, therefore, partially preserving cardiac function. Third, we suggest that PRF scaffold might enhance angiogenesis and neovascularization through the release of pro-angiogenetic factors. This proposal is supported by the findings of augmented angiogenesis activities, including the upregulated expressions of angiogenesis factors and the increased number of small vessels and endothelial progenitor cells in AMI animals treated with ADMSC-embedded PRF scaffold than in those treated by direct ADMSC implantation.

C.-K. Sun et al. / International Journal of Cardiology 173 (2014) 410–423 Fig. 7. Protein expressions of fibrotic, anti-fibrotic, apoptotic and anti-apoptotic biomarkers in infarct area of left ventricular (LV) myocardium on post-infarct day 42 (n = 7). A) The protein expression of transforming growth factor (TGF-β). * vs. other groups with different symbols (*, †, ‡, §), p b 0.006. B) The protein expression of Smad3. * vs. other groups with different symbols (*, †, ‡, §), p b 0.005. C) The protein expression of Smad1/5. * vs. other groups with different symbols (*, †, ‡, §), p b 0.001. D) The protein expression of bone morphogenetic protein (BMP)-2. * vs. other groups with different symbols (*, †, ‡, §), p b 0.001. E) The protein expression of mitochondrial (Mito) Bax. * vs. other groups with different symbols (*, †, ‡, §), p b 0.005. F) The protein expression of cleaved poly(ADP-ribose) polymerase (c-PARP). * vs. other groups with different symbols (*, †, ‡, §), p b 0.006. G) The protein expression of Bcl-2. * vs. other groups with different symbols (*, †, ‡, §), p b 0.005. Statistical analysis using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; ADMSC = adipose-derived mesenchymal stem cell; PRF = platelet-rich fibrin.

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Fig. 8. Protein expressions of heart failure and cardiac hypertrophic biomarkers in infarct area of left ventricular (LV) myocardium on post-infarct day 42 (n = 7). A) The protein expression of brain natriuretic peptide (BNP). * vs. other groups with different symbols (*, †, ‡), p b 0.003. B) The protein expression of β-myosin heavy chain (MHC). * vs. other groups with different symbols (*, †, ‡, §), p b 0.006. C) The protein expression of α-MHC. * vs. other groups with different symbols (*, †, ‡), p b 0.01. Statistical analysis using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; ADMSC = adiposederived mesenchymal stem cell; PRF = platelet-rich fibrin.

4.7. Study limitations This study has limitations. First, although the results of the present study are promising, the exact underlying mechanisms by which ADMSC treatment improves cardiac function remain uncertain. The

proposed mechanisms underlying the improvement of heart function after PRF patching with and without ADMSCs has been schematized in Fig. 11. In conclusion, the results of our study demonstrated that, as compared with the conventional approach of direct ADMSC implantation,

Fig. 9. The protein expressions of biomarkers for angiogenesis and endothelial integrity in infarct area of left ventricular (LV) myocardium on post-infarct day 42 (n = 7). A) The protein expression of CXCR4. * vs. other groups with different symbols (*, †, ‡, §), p b 0.006. B) The protein expression of stromal cell-derived factor (SDF)-1α. * vs. other groups with different symbols (*, †, ‡, §), p b 0.006. C) The protein expression of vascular endothelial growth factor (VEGF). * vs. other groups with different symbols (*, †, ‡, §), p b 0.003. E) The protein expression of endothelial nitric oxide synthase (eNOS). * vs. other groups with different symbols (*, †, ‡, §), p b 0.008. Statistical analysis using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). Symbols (*, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; ADMSC = adipose-derived mesenchymal stem cell; PRF = platelet-rich fibrin.

C.-K. Sun et al. / International Journal of Cardiology 173 (2014) 410–423 Fig. 10. Immunofluorescent (IF) and immunohistochemical (IHC) staining for identification of Inflammatory cells in infarct area of left ventricular (LV) myocardium on post-infarct day 42 (n = 7). A to D) IHC microscopic (400×) identification of CD3+ cells (black arrows). Scale bars in right lower corners represent 20 μm. E) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. F to I) IHC microscopic (400×) identification of CD40+ cells (black arrows). Scale bars in right lower corners represent 20 μm. J) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. K to N) IF microscopic (200×) identification of CD68+ cells (green color); red color indicated implanted ADMSCs. Scale bars in right lower corners represent 50 μm. O) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. P to S) IF microscopic (200×) identification of CD19+ cells (green color); red color indicated implanted ADMSCs. Scale bars in right lower corners represent 50 μm. T) * vs. other groups with different symbols (*, †, ‡, §), p b 0.0001. HPF = high-power field. Statistical analysis using one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). SC = sham control; AMI = acute myocardial infarction; ADMSC = adipose-derived mesenchymal stem cell; PRF = platelet-rich fibrin.

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Fig. 11. Proposed mechanisms underlying the therapeutic effects of platelet-rich fibrin (PRF)-embedded adipose-derived mesenchymal stem cells (ADMSCs) versus direct myocardial ADMSC implantation against acute myocardial infarction-induced left ventricular (LV) dysfunction and remodeling in a rat model. ADMSC = adipose-derived mesenchymal stem cell; PRF = platelet-rich fibrin; BNP = brain natriuretic peptide; TGF = transforming growth factor; c-PARP = cleaved poly(ADP-ribose) polymerase; IL = interleukin; MMP = matrix metalloproteinase; SDF = stromal cell-derived factor; LVEDD = left ventricular end-diastolic dimension; LVEDV = left ventricular end-diastolic volume; LVESD = left ventricular end-systolic dimension; LVESV = left ventricular end-systolic volume; IVS = inter-ventricular septum; MHC = myosin heavy chain; LVFS = left ventricular fractional shortening; LVEF = left ventricular ejection fraction.

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Direct implantation versus platelet-rich fibrin-embedded adipose-derived mesenchymal stem cells in treating rat acute myocardial infarction.

This study tested whether adipose-derived mesenchymal stem cells (ADMSC) embedded in platelet-rich fibrin (PRF) scaffold is superior to direct ADMSC i...
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