- Email: [email protected]
Stem cell-loaded nanofibrous patch promotes the regeneration of infarcted myocardium with functional improvement in rat model
Acta Biomaterialia 10 (2014) 2727–2738
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Stem cell-loaded nanofibrous patch promotes the regeneration of infarcted myocardium with functional improvement in rat model Dan Kai a,c,1, Qiang-Li Wang b,1, Hai-Jie Wang b, Molamma P. Prabhakaran d, Yanzhong Zhang a, Yu-Zhen Tan b,⇑, Seeram Ramakrishna a,c,d,e,⇑ a
College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, China Department of Anatomy, Histology and Embryology, Shanghai Medical School of Fudan University, 138 Yixueyuan Rd, Shanghai 200032, China NUS Graduate School for Integrative Sciences & Engineering, National University of Singapore, Singapore d Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 2 Engineering Drive 3, Singapore e Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore b c
a r t i c l e
i n f o
Article history: Received 28 October 2013 Received in revised form 11 February 2014 Accepted 14 February 2014 Available online 24 February 2014 Keywords: Cardiac patch Electrospun nanofibers Mesenchymal stem cells Myocardial infarction Improvement of cardiac function
a b s t r a c t Myocardial infarction (MI) leads to the loss of cardiomyocytes, followed by left ventricular (LV) remodeling and cardiac dysfunction. The authors hypothesize that an elastic, biodegradable nanofibrous cardiac patch loaded with mesenchymal stem cells (MSC) could restrain LV remodeling and improve cardiac function after MI. Poly(e-caprolactone)/gelatin (PG) nanofibers were fabricated by electrospinning, and the nanofibers displayed a porous and uniform nanofibrous structure with a diameter of 244 ± 51 nm. An MI model was established by ligation of the left anterior descending coronary artery of female Sprague–Dawley rats. The PG nanofibrous patch seeded with MSC, isolated from rat bone marrow, was implanted on the epicardium of the infarcted region of the LV wall of the heart. After transplantation, the PG–cell patch restricted the expansion of the LV wall effectively and reduced the scar size, and the density of the microvessels increased. Cells within the patch were able to migrate towards the scar tissue, and promoted new blood vessel formation at the infarct site. Angiogenesis and the cardiac functions improved significantly after 4 weeks of implantation. The MSC-seeded PG nanofibrous patches are demonstrated to provide sufficient mechanical support, to induce angiogenesis and to accelerate cardiac repair in a rat model of MI. The study highlights the positive impact of implantation of an MSC-seeded PG nanofibrous patch as a novel constituent for MI repair. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Myocardial infarction (MI), a main cause of heart failure, results in the progressive loss of cardiomyocytes and enlargement of the left ventricular (LV) cavity, thus impairing cardiac functions [1,2]. Reversal of MI would require replacement of the lost cardiomyocytes as well as the restoration of the dilated LV wall and blood flow [3]. Traditional cell therapy has shown some positive results for improvement in cardiac performance, but possesses poor cell retention, while transplantation survival plagues this technique [4]. In order to improve the efficiency of cell therapy, a combination of cells within a biomaterial scaffold could be an interesting approach
⇑ Corresponding authors. Address: Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 2 Engineering Drive 3, Singapore (S. Ramakrishna). E-mail addresses: [email protected] (Y.-Z. Tan), [email protected] (S. Ramakrishna). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.actbio.2014.02.030 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
[5]. Cardiac patches could serve as suitable carriers for delivery of graft cells from the biomaterial scaffold, as the implantation of stem cells seeded on cardiac patches would help to reduce the death rate of the graft cells and maximize the ability for myocardial regeneration [6]. Dacron (polyethylene terephthalate), Gore-Tex (polytetrafluoroethylene), glutaraldehyde-treated bovine pericardium or glutaraldehyde-treated homografts are among the currently available clinically applied patches [7]. However, these scaffolding materials are not viable, causing foreign body encapsulation by the host, even necessitating reoperation to replace the patch [8]. In order to develop a regenerative biodegradable patch with a biodegradation rate comparable with the repair process, it is necessary to have a cardiac patch that is elastic and strong enough to resist damage from the contracting myocardium, and at the same time bioactive enough to promote cell adhesion, growth and functionality [9,10]. Currently, some bioactive and biodegradable cardiac patches, such as collagen, chitosan and silk, have been developed for myocardial regeneration. Although encouraging results for transplanting material alone into experimental infarct models have
2728
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
been reported, there are few studies on the influence of stem-cellseeded scaffolds on cardiac repair after MI. Moreover, there is a limited understanding of how the properties of biomimetic scaffolds would affect the outcome of stem cell therapy. In this study, a FDA-approved biodegradable synthetic polymer poly(e-caprolactone) (PCL) and a natural polymer gelatin were blended and electrospun to obtain biomimetic nanofibers as a cardiac patch. Electrospun nanofibrous scaffolds hold great potential and possess many desirable properties for constructing ideal cardiac patches, such as high porosity to permit diffusion of the nutrients and metabolic waste necessary for cell growth, a high surface area to volume ratio to favor cell adhesion, migration, proliferation and differentiation, with controllable fiber diameters to mimic the fibrous architecture of natural extracellular matrix (ECM), and flexibility to formulate multiple polymers and bioactive ingredients with tuneable mechanical properties and biodegradation rate [11,12]. Gelatin was chosen as the natural material, because it is derived from collagen, possesses good biocompatibility and is a relatively low cost material. In order to enhance the mechanical strength of the nanofibrous patch, an elastic polymer, PCL, was incorporated into the system. The characteristics of an electrospun PCL/gelatin (PG) nanofibrous patch loaded with MSC were investigated, and its potential in repairing the infarcted myocardium in rat model was evaluated. MSC isolated from bone marrow were selected in this study because of their capacity for self-renewal and multipotency, well-established isolation/culture technology and low immunogenicity [13]. It was hypothesized that the tissue-engineering strategy using electrospun nanofibrous patches seeded with MSC would result in the formation of functional myocardium that could restrict expansion of the LV wall, promote cardiomyogenesis and angiogenesis, and improve cardiac function after extensive MI. 2. Methods 2.1. Nanofibrous patch preparation and characterization PG (1:1 at weight ratio) was dissolved in 1,1,1,3,3,3-hexafluoro2-propanol to obtain a solution concentration of 7 wt.% by stirring for 24 h. The polymer solution was electrospun from a 5 ml syringe with a 27G blunted stainless steel needle at a flow rate of 1 ml h1. A positive voltage of 12 kV was applied to the needle using a highvoltage power supply (Gamma High Voltage Research, Ormond Beach, FL, USA). An aluminum sheet, used as the collector, was placed at a distance of 10 cm from the needle tip. Nanofibers collected on 15-mm cover slips and aluminum foil were dried overnight under vacuum and used for cell culture and animal experiments. PCL nanofibers as controls were obtained under the same condition. The morphology of the electrospun nanofibers was examined by scanning electron microscopy (SEM) (JSM5600, JEOL, Japan). The diameters of the electrospun fibers were determined from the SEM images, using image analysis software (Image J, National Institutes of Health, Bethesda, VA). The average and standard deviation of the fiber diameter were calculated from 50 random measurements per image. The pore size of the electrospun nanofibers was measured using a capillary flow porometer (CFP-1200-A, Ithaca, NY, USA) by the wet-up/dry-up method. The thicknesses of the membranes were measured by micrometer, and the porosity of the nanofibrous scaffolds was calculated according to the method of Gupta et al. [14]. 2.2. In vitro cell assay All the MSC were isolated from bone marrow of male Sprague– Dawley (SD) rats (12–14 weeks), as previously described [15]. MSC
from the second to the fourth passage were used in the experiments. The nanofibrous sheets of PG and PCL, and glass cover-slips (GC) were placed in a 24-well plate. The nanofibrous sheets were sterilized under UV radiation for 2 h and washed three times with phosphate buffered saline (PBS). Then, MSC were seeded on the sheets or GC at 1.0 104 cells well1. Thereafter, the medium was changed every 3 days. The proliferation of cultured MSC on scaffolds and GC used as controls were evaluated using the colorimetric MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (CellTiter 96 Aqueous One solution, Promega, Madison, WI). After the cells had been cultured for a period of 3, 6 and 9 days, they were rinsed with PBS to remove unattached cells, and incubated with 20% MTS reagent in serumfree medium for a period of 3 h at 37 °C. Absorbance of the dye obtained was measured at 490 nm using a spectrophotometric plate reader (FLUOstar Optima, BMG Lab Technologies, Offenburg, Germany). Cell viability was examined at day 9 after incubation, using the LIVE/DEAD Assay (Molecular Probes, Invitrogen, Singapore). In brief, 20 ll of 2 mM ethidium homodimer solution and 5 ll of 4 mM calcein were added to 10 ml PBS. Each sample was incubated with 1.5 ml of this solution for 1 h at room temperature. After washing, the samples were visualized under confocal laser scanning microscopy (CLSM; Olympus Optical. Co., Ltd., Tokyo, Japan), where live cells produced green fluorescence, while the dead cells showed red fluorescence. The ratios of live cells on GC and nanofibers were calculated using image analysis software, Image J. To compare assembling of the cytoskeleton in cells seeded on different nanofibrous sheets, cells incubated for 9 days were fixed with 2.5% paraformaldehyde for 30 min and permeated using 0.1% Triton X-100. Cells were further incubated with FITC-labeled phalloidin (Sigma) at a dilution of 1:500 in PBS for 90 min and counterstained with 40 ,60 -diamidino-2-phenylindole hydrochloride (DAPI). Samples were then mounted on glass slides, using mounting medium, and imaged with CLSM. 2.3. Establishment of MI model and transplantation of cardiac patches All animals were obtained from the Medical Institute Animal Center of Fudan University (permit number SYXK (Shanghai) 2009-0019), China. The investigation was permitted by the Law of the People’s Republic of China on the Protection of Wildlife and approved by the Institutional Animal Care Committee of Fudan University, China. MI models were established according to the procedure described previously [16]. Forty-two adult female SD rats (250 ± 20 g) were anesthetized with ketamine (60 mg kg1) and xylazine (5–10 mg kg1) by peritoneal injection. Ten rats died after left anterior descending coronary artery (LADCA) ligation, and eight rats died immediately after implantation of cardiac patches (two rats in the PCL group and PG group, respectively; four rats in PG–cell group), as the patch operation took too long in the beginning. The remaining 24 rats were divided into four groups. After endotracheal intubation and ventilation with a rodent ventilator (Taimeng, Chengdu) under anesthetization, the heart was exposed through left lateral thoracotomy and cutting the pericardium open. The LADCA was ligated between the left auricle and conus arteriosus. Successful establishment of MI models was determined by observing a pale discoloration of the myocardium and a high T-wave on the electrocardiogram. After LADCA ligation, the chest of the rats was sutured, and 2 104 U penicillin G benzathine was injected intramuscularly daily for 3 days. Nanofibrous membrane was cut into 0.8 1.0 cm pieces with thickness 100 lm. The nanofibrous sheets were spread on the bottom of culture dish and sterilized under UV radiation for 2 h. Following rinsing in PBS, the sheets were immersed in DMEM
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
2729
overnight. The cells were seeded on the sheets at 2 106 cells per sheet and incubated for 24–36 h, allowing the cells to adhere and spread on the sheets. At 1 week after MI, the rats were randomly divided into control (without any patches), PCL (implanted with PCL nanofibrous sheets), PG (implanted with PG nanofibrous sheets) and PG–cell groups (implanted with MSC-seeded PG nanofibrous sheets). The rats were anesthetized as above, and their hearts were exposed. The infarcted area was covered with the PCL, PG or PG–cell sheet (cell side down for the PG–cell sheet), and the edges of the sheets were sutured on the epicardium of the border of the infarcted myocardium with silk string. In the control group, PBS was injected using a micro-syringe at four spots (20 ll per spot).
room temperature for 1 h. The nucleus was counterstained with DAPI. For each staining, the primary antibodies were displaced with 0.1% BSA as negative control. All images were obtained under fluorescent microscopy (Olympus Corporation, Japan). Microvessel density within the peri-infarct and infarct regions was evaluated by counting positively stained tubular structures in sections in the middle part of infarcted area, and the area of tubular structures was measured with ImagePro Plus 6.0 analysis software (MediaCybernetics). Four high-power fields (20) were randomly selected and analyzed in each section from three independent sections (12 images per animal).
2.4. Echocardiography
To detect the existence and differentiation of the transplanted cells, the Y chromosome specific gene of the cells was detected by fluorescence in situ hybridization, according to a modified procedure described previously [17,18]. In brief, the sections incubated with CD31, cTnT and Cx43 antibody were immersed in 2 SSC for 30 min. Following dehydration and drying, and digesting in 20 lg ll1 protease K, the sections were immersed in denaturation buffer (70% formamide) at 85 °C for 2 min to denature the fixed chromosome specimens. After the biotin-labeled Y chromosome specific probe (Shan Jing, Shanghai) had been denatured in 70% formamide (pH 7.0), the sections were incubated with the denatured probe at 42 °C overnight. After washing, the sections were incubated with streptavidin-FITC (BioLegend, CA) and DyLight 594 AffiniPure goat anti-mouse IgG or streptavidin-cy3 (BioLegend) and FITC AffiniPure goat anti-rabbit IgG for 1 h. The nuclei were counterstained with DAPI. Coexpression of Y chromosome and CD31, cTnT or Cx43 in the transplanted cells was examined by fluorescence microscopy (Olympus Corporation).
Echocardiograms were recorded with an ultrasonocardiograph (Visual Sonics, Toronto) under anesthetization for the rats before MI, at 1 week after MI (and before transplantation, as baseline echocardiogram) and 4 weeks after cardiac patch transplantation. After adequate two-dimensional images had been obtained, the M-mode cursor was positioned to the parasternal long axis view at the level papillary muscles. The LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) were measured from at least three consecutive cardiac cycles. To examine the systolic function, the LV end-diastolic volume (LVEDV), the LV end-systolic volume (LVESV), the ejection fraction (EF = LVEDVLVESV/LVEDV 100%) and fractional shortening (FS = LVEDDLVESD/LVEDD 100%) were measured [16,17]. Improvement in cardiac function was evaluated from changes in the echocardiogram. 2.5. Examination of cardiac tissue All hearts were harvested after echocardiographic analysis at 4 weeks after transplantation of the cardiac patches. The rats were anesthetized with 10% chloral hydrate (3 ml kg1) by peritoneal injection. Following perfusion with 0.9% saline solution, the hearts were fixed with 4% paraformaldehyde for 30 min. Afterwards, the whole heart was removed. With the short axis section, the cardiac wall at the infarcted area was cut into upper, middle and lower parts and continued to be fixed with the above solution for 4 h. After washing, the masses were dehydrated in 20% and 30% sucrose, respectively, and then embedded in Tissue-Tek OCT Compound (Sakura Finetek, CA). Cryostat sections 5 lm thick were prepared. Discontinuous sections obtained from each model were stained with HE and Masson’s trichrome (Sigma), respectively. The change in thickness of the LV wall was evaluated by comparing the thickness of the ventricular wall in three different regions (normal myocardial region, peri-infarcted region and infarcted region). Blue regions in Masson’s stain sections indicate scar tissue. The scar area was defined as the percentage of blue region circumference in whole LV wall circumference in the cross-sections of upper, middle and lower parts of the infarcted region, using computerbased planimetry with ImagePro Plus 6.0 analysis software (MediaCybernetics, Bethesda, MD). 2.6. Immunofluorescent staining To determine angiogenesis in the infarcted tissues and the differentiation of transplanted cells toward endothelial cells and cardiomyocytes, immunostaining of CD31, cardiac troponin T (cTnT) and connexin-43 (Cx43) were performed. The cryostat sections were incubated with primary antibody (1:200, Abcam) at 4 °C overnight. After washing, the sections were incubated with DyLight 594 AffiniPure Goat Anti-Mouse IgG (1:300, EarthOx, CA) or/and FITC AffiniPure Goat Anti-Rabbit IgG (1:50, EarthOx) at
2.7. In situ hybridization
2.8. Statistical analysis All the data presented are expressed as mean ± standard deviation of the mean. Student’s t-test and one-way ANOVA were used, and differences between the groups are considered statistically significant at p < 0.05. 3. Results 3.1. Survival and proliferation of MSC on nanofibrous membrane PG nanofibrous membrane displayed porous and uniform nanofibrous structures, and the fiber diameter of PG nanofibers (244 ± 51 nm) was smaller than that of the PCL nanofibers (882 ± 238 nm) (Fig. 1A). In addition, the structural pore properties of the electrospun nanofibers assessed by capillary flow porometer showed that PCL nanofibers possessed a porosity of 82.4 ± 3.3% with pore size 1.44 ± 0.51 lm, while PG nanofibers had a similar porosity of 83.6 ± 0.8% with a smaller pore size of 0.83 ± 0.15 lm. Rat MSC showed a fibroblast-like morphology. In order to characterize the growth of MSC on nanofibers in vitro, the cell proliferation was evaluated over a span of 9 days. Proliferation of MSC cultured on different nanofibers was analyzed by MTS assay after culturing for 3, 6 and 9 days. Fig. 1B shows that the cell growth on PG nanofibers was statistically significantly higher (p < 0.05) compared with that on PCL nanofibers devoid of gelatin after 9 days of culture. The fluorescent cell viability assay showed that the ratio of cells that survived on PG nanofibers (91.3 ± 3.4%) was higher than that on GC (71.6 ± 4.8%) and PCL (72.5 ± 5.6%) nanofibers after 9 days of culture, while the number of apoptotic and necrotic cells had obviously decreased compared with those on GC and PCL nanofibers (Fig. 1C).
2730
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
Fig. 1. (A) SEM micrographs of electrospun PCL and PG nanofibers. Scale bar = 20 lm. (B) Proliferation of rat MSC on GC, PCL and PG, as determined by MTS assay. ⁄p 6 0.05 indicates statistically significant difference. (C) Live/dead cell assay showing MSC on GC, PCL and PG after 9 days of culture. Live cells show green fluorescence, and dead cells show red fluorescence, and the numbers indicate the ratio of live cells on the substrate. Scale bar = 200 lm.
About 9 days after incubation, the cells seeded on the PG nanofibers were spread well and form an interconnected monolayer, while cells grown on PCL nanofibers remain scattered and poorly aligned (Fig. 2A–C). The cytoskeletal organization of MSC was assessed through immunochemical staining of F-actin. With prominent actin filaments, cells were spread out into different
cytoskeletal architecture on different substrates (Fig. 2D–F). The cells on GC had a flat and thin shape, and were well spread, and the F-actin alignment was observed in parallel arrangement within the cells. In contrast, the cells on PCL nanofibers were poorly spread, showing narrow cell morphology, and the distribution of F-actin in the cells was not uniform. The cells on PG nanofibers
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
2731
Fig. 2. SEM micrographs showing the morphology of rat MSC on (A) GC, (B) PCL and (C) PG, and cytoskeleton assembling (F-actin) of the MSC on (D) GC, (E) PCL and (F) PG after 9 days of culture.
exhibited a well-spread morphology, and there were large numbers of stress fibers in the cells. 3.2. Improvement in cardiac function after patch transplantation A series of echocardiographic examinations were conducted to evaluate the cardiac function of the LV for the study groups. The EF and FS of healthy rats was 79.59 ± 4.94% and 49.31 ± 4.69%, respectively. Cardiac function in all rats was severely compromised by 1 week after LAD occlusion, and decreasing cardiac function continued over the next 4 weeks in the control group. When nanofibrous patches were implanted on the epicardium of the infarcted hearts, the cardiac function of the hearts significantly improved (Fig. 3). The contraction of free wall of the left ventricle was obviously enhanced in the PG–cell group (Fig. 3A). EF (57.40 ± 4.25%) and FS (32.03 ± 1.61%) in the PG–cell group increased significantly compared with baseline and the control, PCL and PG groups (p < 0.01). FS in the PG group was higher than that in the baseline (p < 0.01). EF in the PCL (27.87 ± 4.13%) and PG (35.29 ± 6.88%) groups and FS in the two groups (14.66 ± 2.37% and 20.06 ± 4.51%) also increased significantly compared with the control (EF = 16.15 ± 3.65% and FS = 8.96 ± 2.35%) group (p < 0.01). EF and FS in the PG group were higher than those in the PCL group (p < 0.01). LVEDD, LVESD, LVEDV and LVESV in the PG–cell group were lower than those in the baseline and the control, PCL and PG groups (p < 0.01). In the PCL and PG groups, LVEDD, LVESD, LVEDV and LVESV were obviously decreased compared with the control group (p < 0.01).
and the ventricular cavity enlarged at 5 weeks after MI. In contrast, in the PCL, PG and PG–cell groups, expansion of the free wall of the left ventricle was not observed (Fig. 4A). The ventricular wall of the PG and PG–cell groups was thicker than that of the control and PCL groups, especially in infarcted regions. The thickness of the ventricular wall in the PG–cell group was significantly greater than that in the PG group (p < 0.01) (Fig. 4B, C and E). At 4 weeks after transplantation, some cells were observed in both PCL and PG sheets (Fig. 4B and C). As observed from the Masson’s staining, the fibrous tissue at the infarcted region appeared blue. In the control group, the myocardium of the ventricular wall at the infarcted region was almost substituted by fibrous tissue, and a small myocardium portion was found beneath the endocardium. In the PCL, PG and PG–cell groups, the myocardium was observed under the epicardium. Compared with the PCL group, there was more myocardium in the infarcted region in the PG and PG–cell groups, and the thickness of the myocardium in the PG–cell group was greater than that in the PG group (indicated with M in Fig. 5A). Scar size was defined as the percentage of blue region in the whole wall of the left ventricle in the upper, middle and lower parts of the infarcted region. The scar size of the infarcted region in PCL (41.06 ± 5.47, p < 0.05), PG (20.83 ± 5.48, p < 0.01) and PG– cell sheet (12.96 ± 1.45, p < 0.01) groups was smaller than that in the control group (46.69 ± 3.85). Compared with the PCL group, the scar size in PG and PG–cell groups became significantly smaller (p < 0.01), and the effect of the PG–cell group on cardiac repair was therefore greater than that of the PG group (p < 0.01) (Fig. 5B).
3.3. Morphological changes in left ventricle after patch transplantation
3.4. Expression of cTnT and Cx43 and distribution of Y chromosomepositive cells
In the control group, the free wall of the left ventricle demonstrated thinning, elongation and expansion of the infarcted region,
Cardiomyogenesis in the infarcted region at 4 weeks after transplantation was determined by expression of cTnT and Cx43. For the
2732
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
Fig. 3. Improvement in cardiac function after cardiac patch transplantation. (A) Cardiac function of rats with MI was examined by echocardiography at 4 weeks after transplantation with nanofibrous patches. The contraction of the free wall of the left ventricle was not obviously observed in control, PCL and PG groups. The contraction of free wall of left ventricle in the PG–cell group was significantly improved (arrows). (B) and (C) change in EF and FS, (D) and (E) change in LVEDD and LVESD, (F) and (G) change in LVEDV and LVESV. ⁄p < 0.01 compared with control groups; #p < 0.01 compared with PCL groups; &p < 0.01 compared with PG groups; $p < 0.01 compared with baseline. N = 6.
control group, cTnT was expressed weakly by some cells beneath the endocardium, and Cx43 expression was not observed, while for the PCL group, some cells under the epicardium expressed cTnT and Cx43 weakly. There were more cells expressing cTnT and Cx43 under the epicardium in the PG and PG–cell groups than in the PCL group, and the fluorescence density of cTnT- and Cx43-positive
cells was higher. Cx43 was located mainly at the junction between the cells (Fig. 6A). A Y chromosome probe was used to track the transplanted cells. In situ hybridization in PG–cell sheets demonstrated that Y chromosome-positive cells were distributed mainly on the interior surface of PG, and also in the epicardium and superficial layer of the
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
2733
Fig. 4. Morphological changes in LV wall after 4 weeks of transplantation period. (A) Microphotographs of the transverse sections of whole hearts at the widest parts of the infarcted regions (20). After MI, the LV wall at the infarcted region becomes thinner and expanded (control). In PCL, PG or PG–cell sheet-transplanted heart, the LV wall is thick and does not expand. (B) The peri-infarcted region of the free wall of the left ventricle. The photographs in the second row (bar = 100 lm) are magnification of boxes in the first row (bar = 200 lm). There is myocardium under the epicardium in hearts transplanted with PCL, PG and PG–cell sheets. The myocardium under the epicardium in PG or PG–cell sheet-transplanted heart is thicker than that in PCL sheet-transplanted heart. There is more myocardium under the epicardium in PG–cell sheet-transplanted heart than in PG-transplanted heart. (C) The infarcted region of the free wall of the left ventricle. In PG or PG–cell sheet-transplanted heart, PG is partly degraded. After transplantation, degradation of PCL was not obvious. Asterisks indicate PCL and PG materials. Bar = 100 lm, HE staining. (D) A section of a non-infarcted rat heart was used as reference. (E) Statistical analysis of thickness of the LV wall in infarcted regions (red bars), peri-infarcted regions (green bars) and normal myocardial regions (purple bars). The thickness of the LV wall in infarcted and peri-infarcted regions is larger in PG and PG–cell groups than in the control and PCL groups. Compared with the PG group, the thickness of the LV wall in the PG–cell group is larger. ⁄p < 0.05, #p < 0.01, compared with control group; $p < 0.05, &p < 0.01 compared with PCL group; and –p < 0.05, p < 0.01 compared with PG group. N = 12.
2734
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
Fig. 5. The change in scar size of the infarcted hearts after 4 weeks of transplantation period. (A) Masson’s staining shows the fibrous tissue (blue) and myocardium (M, red) of the LV wall at peri-infarcted (first and second rows) and infarcted regions (third and fourth rows). The photographs in the second and fourth rows are magnifications of the first and third rows, respectively. In the infarcted heart (control), the LV wall at the infarcted region is composed of fibrous tissue; there is only a thin layer of myocardium under the endothelium. In PCL sheet-transplanted heart, dispersed myocardium distributes under the epicardium. In PG or PG–cell sheet-transplanted heart, the myocardium under the epicardium is thick, and there is less fibrous tissue. There is more myocardium under the epicardium in PG–cell sheet-transplanted heart than in PG sheettransplanted heart. There are many microvessels (arrowheads) under the epicardium in PG or PG–cell sheet-transplanted heart. Some microvessels (arrows) are located on the medial surface of PG–cell sheets. V indicates a vessel. Asterisks indicate PCL and PG materials. Bar = 200 lm (first row), 100 lm (second and third rows) or 50 lm (fourth row). (B) Statistical analysis of scar size of the infarcted heart. The scar size is smaller in PCL, PG and PG–cell groups than in the control group. Compared with the PCL group, the scar size of the PG and PG–cell groups is smaller. The scar size of the PG–cell group is smallest in all groups. ⁄p < 0.05, ⁄⁄p < 0.01, compared with control group; #p < 0.01 compared with PCL group; and &p < 0.01 compared with PG group. N = 12.
myocardium. Additionally, some Y chromosome-positive cells were found to express cTnT (Fig. 6B). 3.5. Angiogenesis in the infarcted tissue and MSC differentiation towards endothelial cells Angiogenesis in the peri-infarcted and infarcted regions was determined by evaluating the number of CD31-positive microvessels
at 4 weeks after transplantation. CD31-positive microvessels were observed in the peri-infarcted region and subepicardial tissue of the infarcted region for all groups. There were more microvessels in both the peri-infarcted and infarcted regions in the PG and PG–cell groups. However, the density of the microvessels in the PG–cell group was greater than that in the PG group (Fig. 7A and C). Y chromosome fluorescence in situ hybridization demonstrated that some Y chromosome-positive cells expressed CD31, and some of these
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
2735
Fig. 6. Changes in the cardiac tissue at the infarcted region after transplantation for 4 weeks. The myocardium was identified with expression of cTnT and Cx43. At the infarcted region in the heart without transplantation, there are a few cardiomyocytes (CM) that express cTnT weakly. Cx43 expression in the myocardium was not observed. There is more myocardium at the infarcted region in PCL, PG or PG- cell sheet-transplanted heart. In PCL sheet-transplanted heart, the myocardium expresses cTnT and Cx43 weakly. Expression of cTnT and Cx43 in the myocardium in PG or PG–cell sheet-transplanted heart is obvious. Cx43 distributes at conjunction of between CM. In the merged images, the insets are magnification of small boxes (A). Asterisks indicate PCL and PG materials. Bar = 100 lm. Differentiation of the transplanted cell towards CM (B). The transplanted cells were retraced by Y chromosome fluorescence in situ hybridization and differentiation of these cells towards CM was examined with cTnT immunostaining. In PG–cell sheet-transplanted heart, some Y chromosome-positive cells distribute on the medial surface of PG and under the epicardium, fewer Y chromosome-positive cells express cTnT (arrows). Asterisk indicates PG material. Bar = 100 lm.
cells were located on the wall of the microvessels in the nanofibers of the PG–cell group (Fig. 7B).
4. Discussion An advantage of cardiac patches is that they could restrain the infarcted LV by providing mechanical support to compensate the intraventricular pressure [5]. The requirements for cardiac patches are strict and challenging. An ideal material for cardiac patch should have properties such that the patch should be sufficiently elastic and strong enough to withstand the force of repeated contraction in the heart. PCL is a suitable biomaterial for application in mechanically dynamic environments such as the heart. Besides the mechanical properties, the capability to allow cellular
interactions is also important, and gelatin has appropriate cellular affinity. Therefore, the PG composite nanofibers, as an ECM-mimicking nanofibrous structure, can serve as a cardiac patch possessing appropriate mechanical properties, and could improve the cellular interaction required for efficient tissue regeneration. The mechanical properties of cardiac patches play an important role in preventing chamber dilation. A previous study demonstrated that the Young’s modulus of PG nanofibers (1.45 ± 0.20 MPa) was similar to the stiffness of native myocardium (10–20 kPa at the beginning of diastole and 200–500 kPa at the end of diastole), and a suitable mechanical property of PG nanofibers could reduce the wall stress to allow for the stretching of the myofibers during diastole, resulting in the enhancement of cardiac function [9,19]. The present results show that implantation of PG nanofibers could restrain the expansion of LV effectively and retain cardiac function.
2736
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
Fig. 7. Increase in microvessels at the peri-infarcted (PIR) and infarcted regions (IR) after transplantation for 4 weeks. The microvessels were examined with CD31 immunostaining, distribution of the transplanted cells (arrowheads) were traced with Y chromosome fluorescence in situ hybridization. In all hearts, CD31-positive microvessels can be observed in the peri-infarcted region and under the epicardium (Epi) of the infarcted regions. There are fewer microvessels in the heart without transplantation. There were more microvessels in both the peri-infarcted region and infarcted region in PG or PG–cell sheet-transplanted heart compared with heart without transplantation and PCL sheet-transplanted heart. There are more microvessels in PG–cell sheet-transplanted heart than in PG sheet-transplanted heart (A). Bar = 100 lm. Some Y chromosome-positive cells express CD31 (arrows) at the peri-infarcted region in PG–cell sheet-transplanted heart (B). A few Y chromosome-positive cells expressing CD31 (arrows) are located on the wall of the microvessel in b1. Arrowheads indicate the Y chromosome-positive cells. The large red inset is a magnification of the small red box in b1. b2 is a magnification of the white box in b1. There are microvessels in PG nanofibers, as shown in b2. Asterisks indicate PCL and PG material. Epi, epicardium. Bar = 50 lm in b1. Bar = 25 lm in b2. Statistical analysis of microvessel density in the peri-infarcted and infarcted regions.
Similarly, studies by Fujimoto et al. [20] indicated that polyester urethane urea patches with initial modulus of 1.57 MPa could increase the percentage of fractional area change (%FAC) and maintain the end-diastolic LV cavity area. Cardiac patches traditionally act as a mechanical support to prevent cardiac dilation, but showed limited effects on vascularization or cardiac regeneration after MI [21,22]. Kellar et al. [23] reported that the non-viable cardiac patch (without cells) did not
promote any angiogenesis within infarcted myocardium. However, the present results demonstrate the ability of nanofibrous cardiac patches to trigger new vessel formation, and Fig. 7 shows that the microvessel density in the peri-infarct and infarct regions increased in the PG group compared with the control group, indicating that nanofibrous patches promoted angiogenesis by provoking the endogenous mechanism of vessel formation. One explanation might be that the mechanical support provided by the nanofibrous
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
cardiac patches caused prominent expression of basic fibroblast growth factor and vascular endothelial growth factor in the infarcted myocardium under paracrine mechanisms [24]. Also, the addition of gelatin in PG nanofibers could provide a better biological microenvironment for tissue regeneration. Gelatin contains the arginine-glycine-asparagine tripeptide motif, which has been shown to promote cell anchorage, and it also possess angiogenesis properties [25,26]. The present in vitro results demonstrate that MSC have favorable cell–cell and cell–matrix interaction on PG nanofibers, suggesting the greater advantages of using gelatin along with PCL for MI treatment. The PG cardiac patch with superior performance was used further to deliver cells to infarcted cardiac tissue because of its suitable stiffness and bioactive properties. Implantation of cardiac patches with stem cells could achieve better results in cardiac function. Studies carried out by Chi et al. [27] showed that the implantation of silk fibroin/hyaluronic acid cardiac patches with bone marrow MSC significantly improved the thickness of the LV wall and increased cardiac function compared with the patch devoid of cells. Simpson et al. [28] reported that the implantation of MSC-loaded collagen patches reduced LVESD and increased the FS, whereas the implantation of collagen patches showed no improvement in cardiac function. Compared with those studies, the present results demonstrate that the implantation of MSC-loaded PG not only provided mechanical support to prevent LV dilation and improve the cardiac functions significantly, but also reduced the scar size, increased microvessel density and promoted cardiomyogenesis. In this case, PG nanofibers provided a temporary support for survival and differentiation of the transplanted cells by providing an adhering surface, while the MSC contributed to angiogenesis of infarcted tissues by releasing angiogenic factors and directly participating in microvessel formation. Cardiomyogenesis might be an important procedure towards the improvement of the cardiac function. Studies have reported that the transplanted bone marrow-derived cardiac stem cells or MSC in infarcted hearts could differentiate towards cardiogenic lineage and expressed cardiac specific proteins [29,30]. In the present study, Y chromosome-positive cells expressed cardiacspecific protein cTnT, indicating that those grafted cells could undergo cardiomyogenic differentiation. Another attractive cardiomyogenesis tendency might also be accomplished by the endogenous mechanism after patch implantation. The cardiac patches implanted on the epicardium may activate the endogenous stem cells or the cells in the epicardium, and induce these cells to differentiate into cardiomyocytes and endothelial cells. Accumulating studies also support the hypothesis that the release of stem cellderived paracrine factors could be the predominant mechanisms driving angiogenesis and arteriogenesis in the ischemic myocardium for cardiac recovery. MSC, in particular, could secrete high levels of proangiogenic and proarteriogenic factors that assist in the meditation of neovascularisation [31]. After implantation of a bioengineered cardiac patch with multilayered MSC for myocardial repair in a rat model, Wei et al. [32] observed neo-muscle fibers and neo-microvessels in the cardiac patch with higher expressions of angiogenic cytokines. The present results show that the microvessel density of the infarcted region increased in the PG–cell group compared with the PG group, and Y chromosome-positive cells expressing CD31 were observed in the PG nanofibers and on the wall of some microvessels (Fig. 7), indicating that the MSC might differentiate into endothelial cells and participate in angiogenesis in the infarct myocardium.
5. Conclusion The present study demonstrated the feasibility of the application of electrospun PG nanofibers loaded with MSC as a cardiac
2737
patch, whereby the implanted cardiac patch attenuated LV remodeling and improved cardiac function in a rat infarct model. PG nanofibrous patches served as a mechanical barrier against progressive expansion of the LV wall, and worked together with the grafted MSC to result in cardiomyogenesis and angiogenesis in the infarcted areas. The nanofibrous PG patch loaded with MSC holds great potential as an attractive alternative to cellular cardiomyoplasty or the use of a ventricular restraint device for MI treatment. Acknowledgement This research was supported by Changjiang Scholars Program of the Ministry of Education of China (101-07-005707), NRF-Technion Grant (WBS No: R-398-001-065-592), the National Natural Science Foundation of China (30971674, 81270200) and the Science and Technology Commission of Shanghai Municipality (11540702500). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–7, is difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2014.02. 030. References [1] Zimmermann WH, Eschenhagen T. Cardiac tissue engineering for replacement therapy. Heart Fail Rev 2003;8:259–69. [2] Huang CC, Liao CK, Yang MJ, Chen CH, Hwang SM, Hung YW, et al. A strategy for fabrication of a three-dimensional tissue construct containing uniformly distributed embryoid body-derived cells as a cardiac patch. Biomaterials 2010;31:6218–27. [3] Weissberg P, Qasim A. Stem cell therapy for myocardial repair. Br Med J 2005;91:696. [4] Wang H, Zhou J, Liu Z, Wang C. Injectable cardiac tissue engineering for the treatment of myocardial infarction. J Cell Mol Med 2011;14:1044–55. [5] Wang F, Guan JJ. Cellular cardiomyoplasty and cardiac tissue engineering for myocardial therapy. Adv Drug Delivery Rev 2010;62:784–97. [6] Ota T, Gilbert TW, Badylak SF, Schwartzman D, Zenati MA. Electromechanical characterization of a tissue-engineered myocardial patch derived from extracellular matrix. J Thorac Cardiovasc Surg 2007;133:979–85. [7] Ozawa T, Mickle D, Weisel R, Koyama N, Ozawa S, Li R. Optimal biomaterial for creation of autologous cardiac grafts. Am Heart Assoc 2002:176–82. [8] Chang Y, Chen SC, Wei HJ, Wu TJ, Liang HC, Lai PH, et al. Tissue regeneration observed in a porous acellular bovine pericardium used to repair a myocardial defect in the right ventricle of a rat model. J Thorac Cardiovasc Surg 2005;130:705–11. [9] Chen QZ, Bismarck A, Hansen U, Junaid S, Tran MQ, Harding SE, et al. Characterisation of a soft elastomer poly(glycerol sebacate) designed to match the mechanical properties of myocardial tissue. Biomaterials 2008;29:47–57. [10] Engelmayr G, Cheng M, Bettinger C, Borenstein J, Langer R, Freed L. Accordionlike honeycombs for tissue engineering of cardiac anisotropy. Nat Mater 2008;7:1003–10. [11] Ma K, Chan CK, Liao S, Hwang WYK, Feng Q, Ramakrishna S. Electrospun nanofiber scaffolds for rapid and rich capture of bone marrow-derived hematopoietic stem cells. Biomaterials 2008;29:2096–103. [12] Kai D, Jin G, Prabhakaran MP, Ramakrishna S. Electrospun synthetic and natural nanofibers for regenerative medicine and stem cell. Biotechnol J 2013;8:59–72. [13] Wang H-J, Tan Y-Z. Methods for assessing effects of Wnt/b-catenin signaling in senescence of mesenchymal stem cells. Methods Mol Biol 2013;976:111–30. [14] Gupta D, Venugopal J, Prabhakaran MP, Dev V, Low S, Choon A, et al. Aligned and random nanofibrous substrate for the in vitro culture of Schwann cells for neural tissue engineering. Acta Biomater 2009;5:2560–9. [15] Zhang DY, Wang HJ, Tan YZ. Wnt/beta-catenin signaling induces the aging of mesenchymal stem cells through the DNA damage response and the p53/p21 pathway. PLoS ONE 2011;6:12. [16] Wu JH, Wang HJ, Tan YZ, Li ZH. Characterization of rat very small embryoniclike stem cells and cardiac repair after cell transplantation for myocardial infarction. Stem Cells Dev 2012;21:1367–79. [17] Guo HD, Wang HJ, Tan YZ, Wu JH. Transplantation of marrow-derived cardiac stem cells carried in fibrin improves cardiac function after myocardial infarction. Tissue Eng A 2011;17:45–58. [18] Hoke A, Redett R, Hameed H, Jari R, Zhou C, Li ZB, et al. Schwann cells express motor and sensory phenotypes that regulate axon regeneration. J Neurosci 2006;26:9646–55.
2738
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
[19] Kai D, Prabhakaran MP, Jin GR, Ramakrishna S. Guided orientation of cardiomyocytes on electrospun aligned nanofibers for cardiac tissue engineering. J Biomed Mater Res, Part B-Appl Biomater 2011;98B:379–86. [20] Fujimoto KL, Tobita K, Guan JJ, Hashizume R, Takanari K, Alfieri CM, et al. Placement of an elastic biodegradable cardiac patch on a subacute infarcted heart leads to cellularization with early developmental cardiomyocyte characteristics. J Card Fail 2012;18:585–95. [21] LaPar D, Kron I, Yang Z. Stem cell therapy for ischemic heart disease: where are we? Curr Opin Organ Transplant 2009;14:79. [22] Joggerst SJ, Hatzopoulos AK. Stem cell therapy for cardiac repair: benefits and barriers. Expert Rev Mol Med 2009;11:19. [23] Kellar RS, Landeen LK, Shepherd BR, Naughton GK, Ratcliffe A, Williams SK. Scaffold-based three-dimensional human fibroblast culture provides a structural matrix that supports angiogenesis in infarcted heart tissue. Circulation 2001;104:2063–8. [24] Fujimoto KL, Tobita K, Merryman WD, Guan JJ, Momoi N, Stolz DB, et al. An elastic, biodegradable cardiac patch induces contractile smooth muscle and improves cardiac remodeling and function in subacute myocardial infarction. J Am Coll Cardiol 2007;49:2292–300. [25] Klotz SA, Smith RL. Gelatin fragments block adherence of candida-albicans to extracellular-matrix proteins. Microbiol-Uk 1995;141:2681–4.
[26] Rosso F, Marino G, Giordano A, Barbarisi M, Parmeggiani D, Barbarisi A. Smart materials as scaffolds for tissue engineering. J Cell Physiol 2005;203:465–70. [27] Chi NH, Yang MC, Chung TW, Chen JY, Chou NK, Wang SS. Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model. Biomaterials 2012;33:5541–51. [28] Simpson D, Liu H, Fan THM, Nerem R, Dudley SC. A tissue engineering approach to progenitor cell delivery results in significant cell engraftment and improved myocardial remodeling. Stem Cells 2007;25:2350–7. [29] Guo HD, Cui GH, Wang HJ, Tan YZ. Transplantation of marrow-derived cardiac stem cells carried in designer self-assembling peptide nanofibers improves cardiac function after myocardial infarction. Biochem Biophys Res Commun 2010;399:42–8. [30] Toma C, Pittenger M, Cahill K, Byrne B, Kessler P. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Am Heart Assoc 2002:93–8. [31] Mirotsou M, Jayawardena TM, Schmeckpeper J, Gnecchi M, Dzau VJ. Paracrine mechanisms of stem cell reparative and regenerative actions in the heart. J Mol Cell Cardiol 2011;50:280–9. [32] Wei H, Chen C, Lee W, Chiu I, Hwang S, Lin W, et al. Bioengineered cardiac patch constructed from multilayered mesenchymal stem cells for myocardial repair. Biomaterials 2008;29:3547–56.
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Stem cell-loaded nanofibrous patch promotes the regeneration of infarcted myocardium with functional improvement in rat model Dan Kai a,c,1, Qiang-Li Wang b,1, Hai-Jie Wang b, Molamma P. Prabhakaran d, Yanzhong Zhang a, Yu-Zhen Tan b,⇑, Seeram Ramakrishna a,c,d,e,⇑ a
College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, China Department of Anatomy, Histology and Embryology, Shanghai Medical School of Fudan University, 138 Yixueyuan Rd, Shanghai 200032, China NUS Graduate School for Integrative Sciences & Engineering, National University of Singapore, Singapore d Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 2 Engineering Drive 3, Singapore e Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore b c
a r t i c l e
i n f o
Article history: Received 28 October 2013 Received in revised form 11 February 2014 Accepted 14 February 2014 Available online 24 February 2014 Keywords: Cardiac patch Electrospun nanofibers Mesenchymal stem cells Myocardial infarction Improvement of cardiac function
a b s t r a c t Myocardial infarction (MI) leads to the loss of cardiomyocytes, followed by left ventricular (LV) remodeling and cardiac dysfunction. The authors hypothesize that an elastic, biodegradable nanofibrous cardiac patch loaded with mesenchymal stem cells (MSC) could restrain LV remodeling and improve cardiac function after MI. Poly(e-caprolactone)/gelatin (PG) nanofibers were fabricated by electrospinning, and the nanofibers displayed a porous and uniform nanofibrous structure with a diameter of 244 ± 51 nm. An MI model was established by ligation of the left anterior descending coronary artery of female Sprague–Dawley rats. The PG nanofibrous patch seeded with MSC, isolated from rat bone marrow, was implanted on the epicardium of the infarcted region of the LV wall of the heart. After transplantation, the PG–cell patch restricted the expansion of the LV wall effectively and reduced the scar size, and the density of the microvessels increased. Cells within the patch were able to migrate towards the scar tissue, and promoted new blood vessel formation at the infarct site. Angiogenesis and the cardiac functions improved significantly after 4 weeks of implantation. The MSC-seeded PG nanofibrous patches are demonstrated to provide sufficient mechanical support, to induce angiogenesis and to accelerate cardiac repair in a rat model of MI. The study highlights the positive impact of implantation of an MSC-seeded PG nanofibrous patch as a novel constituent for MI repair. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Myocardial infarction (MI), a main cause of heart failure, results in the progressive loss of cardiomyocytes and enlargement of the left ventricular (LV) cavity, thus impairing cardiac functions [1,2]. Reversal of MI would require replacement of the lost cardiomyocytes as well as the restoration of the dilated LV wall and blood flow [3]. Traditional cell therapy has shown some positive results for improvement in cardiac performance, but possesses poor cell retention, while transplantation survival plagues this technique [4]. In order to improve the efficiency of cell therapy, a combination of cells within a biomaterial scaffold could be an interesting approach
⇑ Corresponding authors. Address: Center for Nanofibers and Nanotechnology, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore, 2 Engineering Drive 3, Singapore (S. Ramakrishna). E-mail addresses: [email protected] (Y.-Z. Tan), [email protected] (S. Ramakrishna). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.actbio.2014.02.030 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
[5]. Cardiac patches could serve as suitable carriers for delivery of graft cells from the biomaterial scaffold, as the implantation of stem cells seeded on cardiac patches would help to reduce the death rate of the graft cells and maximize the ability for myocardial regeneration [6]. Dacron (polyethylene terephthalate), Gore-Tex (polytetrafluoroethylene), glutaraldehyde-treated bovine pericardium or glutaraldehyde-treated homografts are among the currently available clinically applied patches [7]. However, these scaffolding materials are not viable, causing foreign body encapsulation by the host, even necessitating reoperation to replace the patch [8]. In order to develop a regenerative biodegradable patch with a biodegradation rate comparable with the repair process, it is necessary to have a cardiac patch that is elastic and strong enough to resist damage from the contracting myocardium, and at the same time bioactive enough to promote cell adhesion, growth and functionality [9,10]. Currently, some bioactive and biodegradable cardiac patches, such as collagen, chitosan and silk, have been developed for myocardial regeneration. Although encouraging results for transplanting material alone into experimental infarct models have
2728
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
been reported, there are few studies on the influence of stem-cellseeded scaffolds on cardiac repair after MI. Moreover, there is a limited understanding of how the properties of biomimetic scaffolds would affect the outcome of stem cell therapy. In this study, a FDA-approved biodegradable synthetic polymer poly(e-caprolactone) (PCL) and a natural polymer gelatin were blended and electrospun to obtain biomimetic nanofibers as a cardiac patch. Electrospun nanofibrous scaffolds hold great potential and possess many desirable properties for constructing ideal cardiac patches, such as high porosity to permit diffusion of the nutrients and metabolic waste necessary for cell growth, a high surface area to volume ratio to favor cell adhesion, migration, proliferation and differentiation, with controllable fiber diameters to mimic the fibrous architecture of natural extracellular matrix (ECM), and flexibility to formulate multiple polymers and bioactive ingredients with tuneable mechanical properties and biodegradation rate [11,12]. Gelatin was chosen as the natural material, because it is derived from collagen, possesses good biocompatibility and is a relatively low cost material. In order to enhance the mechanical strength of the nanofibrous patch, an elastic polymer, PCL, was incorporated into the system. The characteristics of an electrospun PCL/gelatin (PG) nanofibrous patch loaded with MSC were investigated, and its potential in repairing the infarcted myocardium in rat model was evaluated. MSC isolated from bone marrow were selected in this study because of their capacity for self-renewal and multipotency, well-established isolation/culture technology and low immunogenicity [13]. It was hypothesized that the tissue-engineering strategy using electrospun nanofibrous patches seeded with MSC would result in the formation of functional myocardium that could restrict expansion of the LV wall, promote cardiomyogenesis and angiogenesis, and improve cardiac function after extensive MI. 2. Methods 2.1. Nanofibrous patch preparation and characterization PG (1:1 at weight ratio) was dissolved in 1,1,1,3,3,3-hexafluoro2-propanol to obtain a solution concentration of 7 wt.% by stirring for 24 h. The polymer solution was electrospun from a 5 ml syringe with a 27G blunted stainless steel needle at a flow rate of 1 ml h1. A positive voltage of 12 kV was applied to the needle using a highvoltage power supply (Gamma High Voltage Research, Ormond Beach, FL, USA). An aluminum sheet, used as the collector, was placed at a distance of 10 cm from the needle tip. Nanofibers collected on 15-mm cover slips and aluminum foil were dried overnight under vacuum and used for cell culture and animal experiments. PCL nanofibers as controls were obtained under the same condition. The morphology of the electrospun nanofibers was examined by scanning electron microscopy (SEM) (JSM5600, JEOL, Japan). The diameters of the electrospun fibers were determined from the SEM images, using image analysis software (Image J, National Institutes of Health, Bethesda, VA). The average and standard deviation of the fiber diameter were calculated from 50 random measurements per image. The pore size of the electrospun nanofibers was measured using a capillary flow porometer (CFP-1200-A, Ithaca, NY, USA) by the wet-up/dry-up method. The thicknesses of the membranes were measured by micrometer, and the porosity of the nanofibrous scaffolds was calculated according to the method of Gupta et al. [14]. 2.2. In vitro cell assay All the MSC were isolated from bone marrow of male Sprague– Dawley (SD) rats (12–14 weeks), as previously described [15]. MSC
from the second to the fourth passage were used in the experiments. The nanofibrous sheets of PG and PCL, and glass cover-slips (GC) were placed in a 24-well plate. The nanofibrous sheets were sterilized under UV radiation for 2 h and washed three times with phosphate buffered saline (PBS). Then, MSC were seeded on the sheets or GC at 1.0 104 cells well1. Thereafter, the medium was changed every 3 days. The proliferation of cultured MSC on scaffolds and GC used as controls were evaluated using the colorimetric MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (CellTiter 96 Aqueous One solution, Promega, Madison, WI). After the cells had been cultured for a period of 3, 6 and 9 days, they were rinsed with PBS to remove unattached cells, and incubated with 20% MTS reagent in serumfree medium for a period of 3 h at 37 °C. Absorbance of the dye obtained was measured at 490 nm using a spectrophotometric plate reader (FLUOstar Optima, BMG Lab Technologies, Offenburg, Germany). Cell viability was examined at day 9 after incubation, using the LIVE/DEAD Assay (Molecular Probes, Invitrogen, Singapore). In brief, 20 ll of 2 mM ethidium homodimer solution and 5 ll of 4 mM calcein were added to 10 ml PBS. Each sample was incubated with 1.5 ml of this solution for 1 h at room temperature. After washing, the samples were visualized under confocal laser scanning microscopy (CLSM; Olympus Optical. Co., Ltd., Tokyo, Japan), where live cells produced green fluorescence, while the dead cells showed red fluorescence. The ratios of live cells on GC and nanofibers were calculated using image analysis software, Image J. To compare assembling of the cytoskeleton in cells seeded on different nanofibrous sheets, cells incubated for 9 days were fixed with 2.5% paraformaldehyde for 30 min and permeated using 0.1% Triton X-100. Cells were further incubated with FITC-labeled phalloidin (Sigma) at a dilution of 1:500 in PBS for 90 min and counterstained with 40 ,60 -diamidino-2-phenylindole hydrochloride (DAPI). Samples were then mounted on glass slides, using mounting medium, and imaged with CLSM. 2.3. Establishment of MI model and transplantation of cardiac patches All animals were obtained from the Medical Institute Animal Center of Fudan University (permit number SYXK (Shanghai) 2009-0019), China. The investigation was permitted by the Law of the People’s Republic of China on the Protection of Wildlife and approved by the Institutional Animal Care Committee of Fudan University, China. MI models were established according to the procedure described previously [16]. Forty-two adult female SD rats (250 ± 20 g) were anesthetized with ketamine (60 mg kg1) and xylazine (5–10 mg kg1) by peritoneal injection. Ten rats died after left anterior descending coronary artery (LADCA) ligation, and eight rats died immediately after implantation of cardiac patches (two rats in the PCL group and PG group, respectively; four rats in PG–cell group), as the patch operation took too long in the beginning. The remaining 24 rats were divided into four groups. After endotracheal intubation and ventilation with a rodent ventilator (Taimeng, Chengdu) under anesthetization, the heart was exposed through left lateral thoracotomy and cutting the pericardium open. The LADCA was ligated between the left auricle and conus arteriosus. Successful establishment of MI models was determined by observing a pale discoloration of the myocardium and a high T-wave on the electrocardiogram. After LADCA ligation, the chest of the rats was sutured, and 2 104 U penicillin G benzathine was injected intramuscularly daily for 3 days. Nanofibrous membrane was cut into 0.8 1.0 cm pieces with thickness 100 lm. The nanofibrous sheets were spread on the bottom of culture dish and sterilized under UV radiation for 2 h. Following rinsing in PBS, the sheets were immersed in DMEM
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
2729
overnight. The cells were seeded on the sheets at 2 106 cells per sheet and incubated for 24–36 h, allowing the cells to adhere and spread on the sheets. At 1 week after MI, the rats were randomly divided into control (without any patches), PCL (implanted with PCL nanofibrous sheets), PG (implanted with PG nanofibrous sheets) and PG–cell groups (implanted with MSC-seeded PG nanofibrous sheets). The rats were anesthetized as above, and their hearts were exposed. The infarcted area was covered with the PCL, PG or PG–cell sheet (cell side down for the PG–cell sheet), and the edges of the sheets were sutured on the epicardium of the border of the infarcted myocardium with silk string. In the control group, PBS was injected using a micro-syringe at four spots (20 ll per spot).
room temperature for 1 h. The nucleus was counterstained with DAPI. For each staining, the primary antibodies were displaced with 0.1% BSA as negative control. All images were obtained under fluorescent microscopy (Olympus Corporation, Japan). Microvessel density within the peri-infarct and infarct regions was evaluated by counting positively stained tubular structures in sections in the middle part of infarcted area, and the area of tubular structures was measured with ImagePro Plus 6.0 analysis software (MediaCybernetics). Four high-power fields (20) were randomly selected and analyzed in each section from three independent sections (12 images per animal).
2.4. Echocardiography
To detect the existence and differentiation of the transplanted cells, the Y chromosome specific gene of the cells was detected by fluorescence in situ hybridization, according to a modified procedure described previously [17,18]. In brief, the sections incubated with CD31, cTnT and Cx43 antibody were immersed in 2 SSC for 30 min. Following dehydration and drying, and digesting in 20 lg ll1 protease K, the sections were immersed in denaturation buffer (70% formamide) at 85 °C for 2 min to denature the fixed chromosome specimens. After the biotin-labeled Y chromosome specific probe (Shan Jing, Shanghai) had been denatured in 70% formamide (pH 7.0), the sections were incubated with the denatured probe at 42 °C overnight. After washing, the sections were incubated with streptavidin-FITC (BioLegend, CA) and DyLight 594 AffiniPure goat anti-mouse IgG or streptavidin-cy3 (BioLegend) and FITC AffiniPure goat anti-rabbit IgG for 1 h. The nuclei were counterstained with DAPI. Coexpression of Y chromosome and CD31, cTnT or Cx43 in the transplanted cells was examined by fluorescence microscopy (Olympus Corporation).
Echocardiograms were recorded with an ultrasonocardiograph (Visual Sonics, Toronto) under anesthetization for the rats before MI, at 1 week after MI (and before transplantation, as baseline echocardiogram) and 4 weeks after cardiac patch transplantation. After adequate two-dimensional images had been obtained, the M-mode cursor was positioned to the parasternal long axis view at the level papillary muscles. The LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) were measured from at least three consecutive cardiac cycles. To examine the systolic function, the LV end-diastolic volume (LVEDV), the LV end-systolic volume (LVESV), the ejection fraction (EF = LVEDVLVESV/LVEDV 100%) and fractional shortening (FS = LVEDDLVESD/LVEDD 100%) were measured [16,17]. Improvement in cardiac function was evaluated from changes in the echocardiogram. 2.5. Examination of cardiac tissue All hearts were harvested after echocardiographic analysis at 4 weeks after transplantation of the cardiac patches. The rats were anesthetized with 10% chloral hydrate (3 ml kg1) by peritoneal injection. Following perfusion with 0.9% saline solution, the hearts were fixed with 4% paraformaldehyde for 30 min. Afterwards, the whole heart was removed. With the short axis section, the cardiac wall at the infarcted area was cut into upper, middle and lower parts and continued to be fixed with the above solution for 4 h. After washing, the masses were dehydrated in 20% and 30% sucrose, respectively, and then embedded in Tissue-Tek OCT Compound (Sakura Finetek, CA). Cryostat sections 5 lm thick were prepared. Discontinuous sections obtained from each model were stained with HE and Masson’s trichrome (Sigma), respectively. The change in thickness of the LV wall was evaluated by comparing the thickness of the ventricular wall in three different regions (normal myocardial region, peri-infarcted region and infarcted region). Blue regions in Masson’s stain sections indicate scar tissue. The scar area was defined as the percentage of blue region circumference in whole LV wall circumference in the cross-sections of upper, middle and lower parts of the infarcted region, using computerbased planimetry with ImagePro Plus 6.0 analysis software (MediaCybernetics, Bethesda, MD). 2.6. Immunofluorescent staining To determine angiogenesis in the infarcted tissues and the differentiation of transplanted cells toward endothelial cells and cardiomyocytes, immunostaining of CD31, cardiac troponin T (cTnT) and connexin-43 (Cx43) were performed. The cryostat sections were incubated with primary antibody (1:200, Abcam) at 4 °C overnight. After washing, the sections were incubated with DyLight 594 AffiniPure Goat Anti-Mouse IgG (1:300, EarthOx, CA) or/and FITC AffiniPure Goat Anti-Rabbit IgG (1:50, EarthOx) at
2.7. In situ hybridization
2.8. Statistical analysis All the data presented are expressed as mean ± standard deviation of the mean. Student’s t-test and one-way ANOVA were used, and differences between the groups are considered statistically significant at p < 0.05. 3. Results 3.1. Survival and proliferation of MSC on nanofibrous membrane PG nanofibrous membrane displayed porous and uniform nanofibrous structures, and the fiber diameter of PG nanofibers (244 ± 51 nm) was smaller than that of the PCL nanofibers (882 ± 238 nm) (Fig. 1A). In addition, the structural pore properties of the electrospun nanofibers assessed by capillary flow porometer showed that PCL nanofibers possessed a porosity of 82.4 ± 3.3% with pore size 1.44 ± 0.51 lm, while PG nanofibers had a similar porosity of 83.6 ± 0.8% with a smaller pore size of 0.83 ± 0.15 lm. Rat MSC showed a fibroblast-like morphology. In order to characterize the growth of MSC on nanofibers in vitro, the cell proliferation was evaluated over a span of 9 days. Proliferation of MSC cultured on different nanofibers was analyzed by MTS assay after culturing for 3, 6 and 9 days. Fig. 1B shows that the cell growth on PG nanofibers was statistically significantly higher (p < 0.05) compared with that on PCL nanofibers devoid of gelatin after 9 days of culture. The fluorescent cell viability assay showed that the ratio of cells that survived on PG nanofibers (91.3 ± 3.4%) was higher than that on GC (71.6 ± 4.8%) and PCL (72.5 ± 5.6%) nanofibers after 9 days of culture, while the number of apoptotic and necrotic cells had obviously decreased compared with those on GC and PCL nanofibers (Fig. 1C).
2730
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
Fig. 1. (A) SEM micrographs of electrospun PCL and PG nanofibers. Scale bar = 20 lm. (B) Proliferation of rat MSC on GC, PCL and PG, as determined by MTS assay. ⁄p 6 0.05 indicates statistically significant difference. (C) Live/dead cell assay showing MSC on GC, PCL and PG after 9 days of culture. Live cells show green fluorescence, and dead cells show red fluorescence, and the numbers indicate the ratio of live cells on the substrate. Scale bar = 200 lm.
About 9 days after incubation, the cells seeded on the PG nanofibers were spread well and form an interconnected monolayer, while cells grown on PCL nanofibers remain scattered and poorly aligned (Fig. 2A–C). The cytoskeletal organization of MSC was assessed through immunochemical staining of F-actin. With prominent actin filaments, cells were spread out into different
cytoskeletal architecture on different substrates (Fig. 2D–F). The cells on GC had a flat and thin shape, and were well spread, and the F-actin alignment was observed in parallel arrangement within the cells. In contrast, the cells on PCL nanofibers were poorly spread, showing narrow cell morphology, and the distribution of F-actin in the cells was not uniform. The cells on PG nanofibers
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
2731
Fig. 2. SEM micrographs showing the morphology of rat MSC on (A) GC, (B) PCL and (C) PG, and cytoskeleton assembling (F-actin) of the MSC on (D) GC, (E) PCL and (F) PG after 9 days of culture.
exhibited a well-spread morphology, and there were large numbers of stress fibers in the cells. 3.2. Improvement in cardiac function after patch transplantation A series of echocardiographic examinations were conducted to evaluate the cardiac function of the LV for the study groups. The EF and FS of healthy rats was 79.59 ± 4.94% and 49.31 ± 4.69%, respectively. Cardiac function in all rats was severely compromised by 1 week after LAD occlusion, and decreasing cardiac function continued over the next 4 weeks in the control group. When nanofibrous patches were implanted on the epicardium of the infarcted hearts, the cardiac function of the hearts significantly improved (Fig. 3). The contraction of free wall of the left ventricle was obviously enhanced in the PG–cell group (Fig. 3A). EF (57.40 ± 4.25%) and FS (32.03 ± 1.61%) in the PG–cell group increased significantly compared with baseline and the control, PCL and PG groups (p < 0.01). FS in the PG group was higher than that in the baseline (p < 0.01). EF in the PCL (27.87 ± 4.13%) and PG (35.29 ± 6.88%) groups and FS in the two groups (14.66 ± 2.37% and 20.06 ± 4.51%) also increased significantly compared with the control (EF = 16.15 ± 3.65% and FS = 8.96 ± 2.35%) group (p < 0.01). EF and FS in the PG group were higher than those in the PCL group (p < 0.01). LVEDD, LVESD, LVEDV and LVESV in the PG–cell group were lower than those in the baseline and the control, PCL and PG groups (p < 0.01). In the PCL and PG groups, LVEDD, LVESD, LVEDV and LVESV were obviously decreased compared with the control group (p < 0.01).
and the ventricular cavity enlarged at 5 weeks after MI. In contrast, in the PCL, PG and PG–cell groups, expansion of the free wall of the left ventricle was not observed (Fig. 4A). The ventricular wall of the PG and PG–cell groups was thicker than that of the control and PCL groups, especially in infarcted regions. The thickness of the ventricular wall in the PG–cell group was significantly greater than that in the PG group (p < 0.01) (Fig. 4B, C and E). At 4 weeks after transplantation, some cells were observed in both PCL and PG sheets (Fig. 4B and C). As observed from the Masson’s staining, the fibrous tissue at the infarcted region appeared blue. In the control group, the myocardium of the ventricular wall at the infarcted region was almost substituted by fibrous tissue, and a small myocardium portion was found beneath the endocardium. In the PCL, PG and PG–cell groups, the myocardium was observed under the epicardium. Compared with the PCL group, there was more myocardium in the infarcted region in the PG and PG–cell groups, and the thickness of the myocardium in the PG–cell group was greater than that in the PG group (indicated with M in Fig. 5A). Scar size was defined as the percentage of blue region in the whole wall of the left ventricle in the upper, middle and lower parts of the infarcted region. The scar size of the infarcted region in PCL (41.06 ± 5.47, p < 0.05), PG (20.83 ± 5.48, p < 0.01) and PG– cell sheet (12.96 ± 1.45, p < 0.01) groups was smaller than that in the control group (46.69 ± 3.85). Compared with the PCL group, the scar size in PG and PG–cell groups became significantly smaller (p < 0.01), and the effect of the PG–cell group on cardiac repair was therefore greater than that of the PG group (p < 0.01) (Fig. 5B).
3.3. Morphological changes in left ventricle after patch transplantation
3.4. Expression of cTnT and Cx43 and distribution of Y chromosomepositive cells
In the control group, the free wall of the left ventricle demonstrated thinning, elongation and expansion of the infarcted region,
Cardiomyogenesis in the infarcted region at 4 weeks after transplantation was determined by expression of cTnT and Cx43. For the
2732
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
Fig. 3. Improvement in cardiac function after cardiac patch transplantation. (A) Cardiac function of rats with MI was examined by echocardiography at 4 weeks after transplantation with nanofibrous patches. The contraction of the free wall of the left ventricle was not obviously observed in control, PCL and PG groups. The contraction of free wall of left ventricle in the PG–cell group was significantly improved (arrows). (B) and (C) change in EF and FS, (D) and (E) change in LVEDD and LVESD, (F) and (G) change in LVEDV and LVESV. ⁄p < 0.01 compared with control groups; #p < 0.01 compared with PCL groups; &p < 0.01 compared with PG groups; $p < 0.01 compared with baseline. N = 6.
control group, cTnT was expressed weakly by some cells beneath the endocardium, and Cx43 expression was not observed, while for the PCL group, some cells under the epicardium expressed cTnT and Cx43 weakly. There were more cells expressing cTnT and Cx43 under the epicardium in the PG and PG–cell groups than in the PCL group, and the fluorescence density of cTnT- and Cx43-positive
cells was higher. Cx43 was located mainly at the junction between the cells (Fig. 6A). A Y chromosome probe was used to track the transplanted cells. In situ hybridization in PG–cell sheets demonstrated that Y chromosome-positive cells were distributed mainly on the interior surface of PG, and also in the epicardium and superficial layer of the
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
2733
Fig. 4. Morphological changes in LV wall after 4 weeks of transplantation period. (A) Microphotographs of the transverse sections of whole hearts at the widest parts of the infarcted regions (20). After MI, the LV wall at the infarcted region becomes thinner and expanded (control). In PCL, PG or PG–cell sheet-transplanted heart, the LV wall is thick and does not expand. (B) The peri-infarcted region of the free wall of the left ventricle. The photographs in the second row (bar = 100 lm) are magnification of boxes in the first row (bar = 200 lm). There is myocardium under the epicardium in hearts transplanted with PCL, PG and PG–cell sheets. The myocardium under the epicardium in PG or PG–cell sheet-transplanted heart is thicker than that in PCL sheet-transplanted heart. There is more myocardium under the epicardium in PG–cell sheet-transplanted heart than in PG-transplanted heart. (C) The infarcted region of the free wall of the left ventricle. In PG or PG–cell sheet-transplanted heart, PG is partly degraded. After transplantation, degradation of PCL was not obvious. Asterisks indicate PCL and PG materials. Bar = 100 lm, HE staining. (D) A section of a non-infarcted rat heart was used as reference. (E) Statistical analysis of thickness of the LV wall in infarcted regions (red bars), peri-infarcted regions (green bars) and normal myocardial regions (purple bars). The thickness of the LV wall in infarcted and peri-infarcted regions is larger in PG and PG–cell groups than in the control and PCL groups. Compared with the PG group, the thickness of the LV wall in the PG–cell group is larger. ⁄p < 0.05, #p < 0.01, compared with control group; $p < 0.05, &p < 0.01 compared with PCL group; and –p < 0.05, p < 0.01 compared with PG group. N = 12.
2734
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
Fig. 5. The change in scar size of the infarcted hearts after 4 weeks of transplantation period. (A) Masson’s staining shows the fibrous tissue (blue) and myocardium (M, red) of the LV wall at peri-infarcted (first and second rows) and infarcted regions (third and fourth rows). The photographs in the second and fourth rows are magnifications of the first and third rows, respectively. In the infarcted heart (control), the LV wall at the infarcted region is composed of fibrous tissue; there is only a thin layer of myocardium under the endothelium. In PCL sheet-transplanted heart, dispersed myocardium distributes under the epicardium. In PG or PG–cell sheet-transplanted heart, the myocardium under the epicardium is thick, and there is less fibrous tissue. There is more myocardium under the epicardium in PG–cell sheet-transplanted heart than in PG sheettransplanted heart. There are many microvessels (arrowheads) under the epicardium in PG or PG–cell sheet-transplanted heart. Some microvessels (arrows) are located on the medial surface of PG–cell sheets. V indicates a vessel. Asterisks indicate PCL and PG materials. Bar = 200 lm (first row), 100 lm (second and third rows) or 50 lm (fourth row). (B) Statistical analysis of scar size of the infarcted heart. The scar size is smaller in PCL, PG and PG–cell groups than in the control group. Compared with the PCL group, the scar size of the PG and PG–cell groups is smaller. The scar size of the PG–cell group is smallest in all groups. ⁄p < 0.05, ⁄⁄p < 0.01, compared with control group; #p < 0.01 compared with PCL group; and &p < 0.01 compared with PG group. N = 12.
myocardium. Additionally, some Y chromosome-positive cells were found to express cTnT (Fig. 6B). 3.5. Angiogenesis in the infarcted tissue and MSC differentiation towards endothelial cells Angiogenesis in the peri-infarcted and infarcted regions was determined by evaluating the number of CD31-positive microvessels
at 4 weeks after transplantation. CD31-positive microvessels were observed in the peri-infarcted region and subepicardial tissue of the infarcted region for all groups. There were more microvessels in both the peri-infarcted and infarcted regions in the PG and PG–cell groups. However, the density of the microvessels in the PG–cell group was greater than that in the PG group (Fig. 7A and C). Y chromosome fluorescence in situ hybridization demonstrated that some Y chromosome-positive cells expressed CD31, and some of these
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
2735
Fig. 6. Changes in the cardiac tissue at the infarcted region after transplantation for 4 weeks. The myocardium was identified with expression of cTnT and Cx43. At the infarcted region in the heart without transplantation, there are a few cardiomyocytes (CM) that express cTnT weakly. Cx43 expression in the myocardium was not observed. There is more myocardium at the infarcted region in PCL, PG or PG- cell sheet-transplanted heart. In PCL sheet-transplanted heart, the myocardium expresses cTnT and Cx43 weakly. Expression of cTnT and Cx43 in the myocardium in PG or PG–cell sheet-transplanted heart is obvious. Cx43 distributes at conjunction of between CM. In the merged images, the insets are magnification of small boxes (A). Asterisks indicate PCL and PG materials. Bar = 100 lm. Differentiation of the transplanted cell towards CM (B). The transplanted cells were retraced by Y chromosome fluorescence in situ hybridization and differentiation of these cells towards CM was examined with cTnT immunostaining. In PG–cell sheet-transplanted heart, some Y chromosome-positive cells distribute on the medial surface of PG and under the epicardium, fewer Y chromosome-positive cells express cTnT (arrows). Asterisk indicates PG material. Bar = 100 lm.
cells were located on the wall of the microvessels in the nanofibers of the PG–cell group (Fig. 7B).
4. Discussion An advantage of cardiac patches is that they could restrain the infarcted LV by providing mechanical support to compensate the intraventricular pressure [5]. The requirements for cardiac patches are strict and challenging. An ideal material for cardiac patch should have properties such that the patch should be sufficiently elastic and strong enough to withstand the force of repeated contraction in the heart. PCL is a suitable biomaterial for application in mechanically dynamic environments such as the heart. Besides the mechanical properties, the capability to allow cellular
interactions is also important, and gelatin has appropriate cellular affinity. Therefore, the PG composite nanofibers, as an ECM-mimicking nanofibrous structure, can serve as a cardiac patch possessing appropriate mechanical properties, and could improve the cellular interaction required for efficient tissue regeneration. The mechanical properties of cardiac patches play an important role in preventing chamber dilation. A previous study demonstrated that the Young’s modulus of PG nanofibers (1.45 ± 0.20 MPa) was similar to the stiffness of native myocardium (10–20 kPa at the beginning of diastole and 200–500 kPa at the end of diastole), and a suitable mechanical property of PG nanofibers could reduce the wall stress to allow for the stretching of the myofibers during diastole, resulting in the enhancement of cardiac function [9,19]. The present results show that implantation of PG nanofibers could restrain the expansion of LV effectively and retain cardiac function.
2736
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
Fig. 7. Increase in microvessels at the peri-infarcted (PIR) and infarcted regions (IR) after transplantation for 4 weeks. The microvessels were examined with CD31 immunostaining, distribution of the transplanted cells (arrowheads) were traced with Y chromosome fluorescence in situ hybridization. In all hearts, CD31-positive microvessels can be observed in the peri-infarcted region and under the epicardium (Epi) of the infarcted regions. There are fewer microvessels in the heart without transplantation. There were more microvessels in both the peri-infarcted region and infarcted region in PG or PG–cell sheet-transplanted heart compared with heart without transplantation and PCL sheet-transplanted heart. There are more microvessels in PG–cell sheet-transplanted heart than in PG sheet-transplanted heart (A). Bar = 100 lm. Some Y chromosome-positive cells express CD31 (arrows) at the peri-infarcted region in PG–cell sheet-transplanted heart (B). A few Y chromosome-positive cells expressing CD31 (arrows) are located on the wall of the microvessel in b1. Arrowheads indicate the Y chromosome-positive cells. The large red inset is a magnification of the small red box in b1. b2 is a magnification of the white box in b1. There are microvessels in PG nanofibers, as shown in b2. Asterisks indicate PCL and PG material. Epi, epicardium. Bar = 50 lm in b1. Bar = 25 lm in b2. Statistical analysis of microvessel density in the peri-infarcted and infarcted regions.
Similarly, studies by Fujimoto et al. [20] indicated that polyester urethane urea patches with initial modulus of 1.57 MPa could increase the percentage of fractional area change (%FAC) and maintain the end-diastolic LV cavity area. Cardiac patches traditionally act as a mechanical support to prevent cardiac dilation, but showed limited effects on vascularization or cardiac regeneration after MI [21,22]. Kellar et al. [23] reported that the non-viable cardiac patch (without cells) did not
promote any angiogenesis within infarcted myocardium. However, the present results demonstrate the ability of nanofibrous cardiac patches to trigger new vessel formation, and Fig. 7 shows that the microvessel density in the peri-infarct and infarct regions increased in the PG group compared with the control group, indicating that nanofibrous patches promoted angiogenesis by provoking the endogenous mechanism of vessel formation. One explanation might be that the mechanical support provided by the nanofibrous
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
cardiac patches caused prominent expression of basic fibroblast growth factor and vascular endothelial growth factor in the infarcted myocardium under paracrine mechanisms [24]. Also, the addition of gelatin in PG nanofibers could provide a better biological microenvironment for tissue regeneration. Gelatin contains the arginine-glycine-asparagine tripeptide motif, which has been shown to promote cell anchorage, and it also possess angiogenesis properties [25,26]. The present in vitro results demonstrate that MSC have favorable cell–cell and cell–matrix interaction on PG nanofibers, suggesting the greater advantages of using gelatin along with PCL for MI treatment. The PG cardiac patch with superior performance was used further to deliver cells to infarcted cardiac tissue because of its suitable stiffness and bioactive properties. Implantation of cardiac patches with stem cells could achieve better results in cardiac function. Studies carried out by Chi et al. [27] showed that the implantation of silk fibroin/hyaluronic acid cardiac patches with bone marrow MSC significantly improved the thickness of the LV wall and increased cardiac function compared with the patch devoid of cells. Simpson et al. [28] reported that the implantation of MSC-loaded collagen patches reduced LVESD and increased the FS, whereas the implantation of collagen patches showed no improvement in cardiac function. Compared with those studies, the present results demonstrate that the implantation of MSC-loaded PG not only provided mechanical support to prevent LV dilation and improve the cardiac functions significantly, but also reduced the scar size, increased microvessel density and promoted cardiomyogenesis. In this case, PG nanofibers provided a temporary support for survival and differentiation of the transplanted cells by providing an adhering surface, while the MSC contributed to angiogenesis of infarcted tissues by releasing angiogenic factors and directly participating in microvessel formation. Cardiomyogenesis might be an important procedure towards the improvement of the cardiac function. Studies have reported that the transplanted bone marrow-derived cardiac stem cells or MSC in infarcted hearts could differentiate towards cardiogenic lineage and expressed cardiac specific proteins [29,30]. In the present study, Y chromosome-positive cells expressed cardiacspecific protein cTnT, indicating that those grafted cells could undergo cardiomyogenic differentiation. Another attractive cardiomyogenesis tendency might also be accomplished by the endogenous mechanism after patch implantation. The cardiac patches implanted on the epicardium may activate the endogenous stem cells or the cells in the epicardium, and induce these cells to differentiate into cardiomyocytes and endothelial cells. Accumulating studies also support the hypothesis that the release of stem cellderived paracrine factors could be the predominant mechanisms driving angiogenesis and arteriogenesis in the ischemic myocardium for cardiac recovery. MSC, in particular, could secrete high levels of proangiogenic and proarteriogenic factors that assist in the meditation of neovascularisation [31]. After implantation of a bioengineered cardiac patch with multilayered MSC for myocardial repair in a rat model, Wei et al. [32] observed neo-muscle fibers and neo-microvessels in the cardiac patch with higher expressions of angiogenic cytokines. The present results show that the microvessel density of the infarcted region increased in the PG–cell group compared with the PG group, and Y chromosome-positive cells expressing CD31 were observed in the PG nanofibers and on the wall of some microvessels (Fig. 7), indicating that the MSC might differentiate into endothelial cells and participate in angiogenesis in the infarct myocardium.
5. Conclusion The present study demonstrated the feasibility of the application of electrospun PG nanofibers loaded with MSC as a cardiac
2737
patch, whereby the implanted cardiac patch attenuated LV remodeling and improved cardiac function in a rat infarct model. PG nanofibrous patches served as a mechanical barrier against progressive expansion of the LV wall, and worked together with the grafted MSC to result in cardiomyogenesis and angiogenesis in the infarcted areas. The nanofibrous PG patch loaded with MSC holds great potential as an attractive alternative to cellular cardiomyoplasty or the use of a ventricular restraint device for MI treatment. Acknowledgement This research was supported by Changjiang Scholars Program of the Ministry of Education of China (101-07-005707), NRF-Technion Grant (WBS No: R-398-001-065-592), the National Natural Science Foundation of China (30971674, 81270200) and the Science and Technology Commission of Shanghai Municipality (11540702500). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–7, is difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2014.02. 030. References [1] Zimmermann WH, Eschenhagen T. Cardiac tissue engineering for replacement therapy. Heart Fail Rev 2003;8:259–69. [2] Huang CC, Liao CK, Yang MJ, Chen CH, Hwang SM, Hung YW, et al. A strategy for fabrication of a three-dimensional tissue construct containing uniformly distributed embryoid body-derived cells as a cardiac patch. Biomaterials 2010;31:6218–27. [3] Weissberg P, Qasim A. Stem cell therapy for myocardial repair. Br Med J 2005;91:696. [4] Wang H, Zhou J, Liu Z, Wang C. Injectable cardiac tissue engineering for the treatment of myocardial infarction. J Cell Mol Med 2011;14:1044–55. [5] Wang F, Guan JJ. Cellular cardiomyoplasty and cardiac tissue engineering for myocardial therapy. Adv Drug Delivery Rev 2010;62:784–97. [6] Ota T, Gilbert TW, Badylak SF, Schwartzman D, Zenati MA. Electromechanical characterization of a tissue-engineered myocardial patch derived from extracellular matrix. J Thorac Cardiovasc Surg 2007;133:979–85. [7] Ozawa T, Mickle D, Weisel R, Koyama N, Ozawa S, Li R. Optimal biomaterial for creation of autologous cardiac grafts. Am Heart Assoc 2002:176–82. [8] Chang Y, Chen SC, Wei HJ, Wu TJ, Liang HC, Lai PH, et al. Tissue regeneration observed in a porous acellular bovine pericardium used to repair a myocardial defect in the right ventricle of a rat model. J Thorac Cardiovasc Surg 2005;130:705–11. [9] Chen QZ, Bismarck A, Hansen U, Junaid S, Tran MQ, Harding SE, et al. Characterisation of a soft elastomer poly(glycerol sebacate) designed to match the mechanical properties of myocardial tissue. Biomaterials 2008;29:47–57. [10] Engelmayr G, Cheng M, Bettinger C, Borenstein J, Langer R, Freed L. Accordionlike honeycombs for tissue engineering of cardiac anisotropy. Nat Mater 2008;7:1003–10. [11] Ma K, Chan CK, Liao S, Hwang WYK, Feng Q, Ramakrishna S. Electrospun nanofiber scaffolds for rapid and rich capture of bone marrow-derived hematopoietic stem cells. Biomaterials 2008;29:2096–103. [12] Kai D, Jin G, Prabhakaran MP, Ramakrishna S. Electrospun synthetic and natural nanofibers for regenerative medicine and stem cell. Biotechnol J 2013;8:59–72. [13] Wang H-J, Tan Y-Z. Methods for assessing effects of Wnt/b-catenin signaling in senescence of mesenchymal stem cells. Methods Mol Biol 2013;976:111–30. [14] Gupta D, Venugopal J, Prabhakaran MP, Dev V, Low S, Choon A, et al. Aligned and random nanofibrous substrate for the in vitro culture of Schwann cells for neural tissue engineering. Acta Biomater 2009;5:2560–9. [15] Zhang DY, Wang HJ, Tan YZ. Wnt/beta-catenin signaling induces the aging of mesenchymal stem cells through the DNA damage response and the p53/p21 pathway. PLoS ONE 2011;6:12. [16] Wu JH, Wang HJ, Tan YZ, Li ZH. Characterization of rat very small embryoniclike stem cells and cardiac repair after cell transplantation for myocardial infarction. Stem Cells Dev 2012;21:1367–79. [17] Guo HD, Wang HJ, Tan YZ, Wu JH. Transplantation of marrow-derived cardiac stem cells carried in fibrin improves cardiac function after myocardial infarction. Tissue Eng A 2011;17:45–58. [18] Hoke A, Redett R, Hameed H, Jari R, Zhou C, Li ZB, et al. Schwann cells express motor and sensory phenotypes that regulate axon regeneration. J Neurosci 2006;26:9646–55.
2738
D. Kai et al. / Acta Biomaterialia 10 (2014) 2727–2738
[19] Kai D, Prabhakaran MP, Jin GR, Ramakrishna S. Guided orientation of cardiomyocytes on electrospun aligned nanofibers for cardiac tissue engineering. J Biomed Mater Res, Part B-Appl Biomater 2011;98B:379–86. [20] Fujimoto KL, Tobita K, Guan JJ, Hashizume R, Takanari K, Alfieri CM, et al. Placement of an elastic biodegradable cardiac patch on a subacute infarcted heart leads to cellularization with early developmental cardiomyocyte characteristics. J Card Fail 2012;18:585–95. [21] LaPar D, Kron I, Yang Z. Stem cell therapy for ischemic heart disease: where are we? Curr Opin Organ Transplant 2009;14:79. [22] Joggerst SJ, Hatzopoulos AK. Stem cell therapy for cardiac repair: benefits and barriers. Expert Rev Mol Med 2009;11:19. [23] Kellar RS, Landeen LK, Shepherd BR, Naughton GK, Ratcliffe A, Williams SK. Scaffold-based three-dimensional human fibroblast culture provides a structural matrix that supports angiogenesis in infarcted heart tissue. Circulation 2001;104:2063–8. [24] Fujimoto KL, Tobita K, Merryman WD, Guan JJ, Momoi N, Stolz DB, et al. An elastic, biodegradable cardiac patch induces contractile smooth muscle and improves cardiac remodeling and function in subacute myocardial infarction. J Am Coll Cardiol 2007;49:2292–300. [25] Klotz SA, Smith RL. Gelatin fragments block adherence of candida-albicans to extracellular-matrix proteins. Microbiol-Uk 1995;141:2681–4.
[26] Rosso F, Marino G, Giordano A, Barbarisi M, Parmeggiani D, Barbarisi A. Smart materials as scaffolds for tissue engineering. J Cell Physiol 2005;203:465–70. [27] Chi NH, Yang MC, Chung TW, Chen JY, Chou NK, Wang SS. Cardiac repair achieved by bone marrow mesenchymal stem cells/silk fibroin/hyaluronic acid patches in a rat of myocardial infarction model. Biomaterials 2012;33:5541–51. [28] Simpson D, Liu H, Fan THM, Nerem R, Dudley SC. A tissue engineering approach to progenitor cell delivery results in significant cell engraftment and improved myocardial remodeling. Stem Cells 2007;25:2350–7. [29] Guo HD, Cui GH, Wang HJ, Tan YZ. Transplantation of marrow-derived cardiac stem cells carried in designer self-assembling peptide nanofibers improves cardiac function after myocardial infarction. Biochem Biophys Res Commun 2010;399:42–8. [30] Toma C, Pittenger M, Cahill K, Byrne B, Kessler P. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Am Heart Assoc 2002:93–8. [31] Mirotsou M, Jayawardena TM, Schmeckpeper J, Gnecchi M, Dzau VJ. Paracrine mechanisms of stem cell reparative and regenerative actions in the heart. J Mol Cell Cardiol 2011;50:280–9. [32] Wei H, Chen C, Lee W, Chiu I, Hwang S, Lin W, et al. Bioengineered cardiac patch constructed from multilayered mesenchymal stem cells for myocardial repair. Biomaterials 2008;29:3547–56.