Materials
For fabrication of the patch and stem cell spheroid, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin antibiotics (P/S), and trypsin were purchased from Gibco BRL (Gaithersburg, MD, USA). Phosphate-buffered saline (PBS) was purchased from Biosesang (Seongnam, Korea). Human adipose-derived stem cell (hADSC) was obtained from Lonza (Walkersville, MD, USA). The biopsy punch was purchased from Kai Medical (Gifu, Japan). For the in vitro spheroid study, TRIzol was purchased from Ambion (Austin, TX, USA). Chloroform, isopropyl alcohol, ethanol, and β-mercaptoethanol were purchased from Sigma-Aldrich (St. Louis, MO, USA). AccuPower® RocketScript™ Cycle RT PreMix and AccuPower® 2X Greenstar™ qPCR MasterMix were purchased from Bioneer (Daejeon, Korea). PRO-PREP™ Protein Extraction Solution was obtained from iNtRON Biotechnology (Seongnam, Korea). Bradford reagent and 4× Laemmli sample buffer were obtained from Bio-Rad (Hercules, CA, USA). 5% skim milk was purchased from BD Difco™ (Detroit, MD, USA). Antibodies of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Bcl-2-associated X (BAX), and hepatocyte growth factor (HGF) were purchased from Abcam (Cambridge, MA, USA). Antibodies against E-cadherin and B-cell lymphoma 2 (BCL-2) were purchased from Cell Signaling Technology (Danvers, MA, USA), and the vascular endothelial growth factor (VEGF) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA). The ECL reagent WESTSAVE UP was purchased from AbFrontier (Seoul, Korea). The X-ray films were obtained from AGFA HealthCare NV (Mortsel, Belgium). The Proteome Profiler Human Angiogenesis Array Kit was purchased from R&D Systems (Minneapolis, MN, USA). For in vivo animal study, 8–10-week-old male Sprague-Dawley (SD) rats were supplied by Orient Bio Inc. (Seongnam, Korea). 6 − 0 polypropylene, 4 − 0 surgifit, and 6 − 0 black silk sutures were obtained from Ailee (Busan, Korea). 10% formalin and paraffin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Xylene was obtained from Daejung (Siheung, Korea). The hematoxylin and eosin (H&E) staining kit was purchased from Abcam (Cambridge, MA, USA), and Masson’s trichrome was purchased from Dako (Hamburg, Germany). A Millipore Milli-Q purification system treated with water was used for all the experiments.
For polymer synthesis and characterization, gelatin (Gtn, type A from porcine skin, < 300 bloom), cystamine dihydrochloride (CYS), 6-maleimidohexanoic acid (MHA), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), DL-dithiothreitol (DTT), and deuterium oxide (D2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s phosphate-buffered saline (DPBS) was obtained from Gibco (Grand Island, NY, USA). Dialysis membranes (molecular cut-off = 3.5 kDa) were supplied by Spectrum Laboratories (Rancho Dominguez, CA, USA). Hydrochloric acid (HCl, 1N) was obtained from Daejung (Siheung, Korea). 5,5’-Dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) was provided by Thermo Scientific (Rockford, IL, USA). For the in vitro cell study, human dermal fibroblast (HDF) was purchased from Lonza (Walkersville, MD, USA). DMEM, newborn calf serum (NBCS), P/S, and 0.25% trypsin-EDTA were supplied by Gibco (Grand Island, NY, USA). The Live/Dead kit was obtained from Invitrogen (Grand Island, NY, USA). The WST-1 cell proliferation kit was purchased from Roche (Basel, Switzerland). For the in vivo animal study, 5-week-old female C57BL mice were obtained from Koatech (Gyeonggi-do, Korea). Isoflurane (Forane Solution) was purchased from Hana Pharm Co. (Seongnam, Korea). Formalin (10%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hematoxylin H and aqueous eosin Y solutions were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Fabrication of open and open/closed pocket patches for stem cell spheroid encapsulation
In our previous study, we developed an elastomeric bioscaffold (TPU-CEC363) with tunable biodegradability and elasticity for noninvasive stem cell-based therapy [59]. In this study, developed elastomer was used as a bioink to fabricate stem cell spheroid-encapsulating patches. Briefly, we synthesized TPU-CEC363 using poly(ethylene glycol) (PEG; Sigma-Aldrich), ε-caprolactone (Tokyo Chemical Industry, Tokyo, Japan), iron (Ⅲ) acetylacetonate (Sigma-Aldrich), and toluene (Daejung). TPU-CEC363 was then synthesized using the tri-block copolymer, toluene, and hexamethylene diisocyanate. As a result, TPU-CEC363 containing 50% each of PEG and polycaprolactone (PCL) was prepared. The synthesized TPU-CEC363 was extruded to a diameter of 1.75 mm, and the 3D patch was 3D printed using FDM Vis Power Plus (Vision Technology Korea, Daejeon, Korea) [60].
Characterization of open and open/closed pocket patches
The 3D-printed patches with a size of 25 × 25 mm were supplied by the Korea Institute of Science and Technology and Vision Technology Korea. The 3D patches were cut using a biopsy punch with a diameter of 4 mm, and the number of pockets in each patch was observed under a light microscope (CKX41, Olympus, Tokyo, Japan). The swelling ability was performed with 1× PBS, and photographs of the pockets were captured using a light microscope (CKX41, Olympus) and Infinity analyze software (Lumenera Corporation, Ottawa, Canada). The pocket size was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Preparation of 3D patch and spheroid formation in 3D patch
The 3D patches with a diameter of 4 mm were placed into each well of a 96-well plate, and the 96-well plate without a lid was placed in a sterilized pack and sterilized with ethylene oxide gas at 37°C for 3 h. A 96-well plate containing sterilized 3D patches was refrigerated and protected from light to slow degradation. Before use, the 3D patch was swollen by adding 1× PBS to the wells of a 96-well plate. The 3D patch in PBS was centrifuged at 1,500 rpm for 5 min, and the PBS was replaced with serum-free DMEM. The hADSCs were cultured with DMEM containing 10% (v/v) FBS and 1% (v/v) P/S in a 5% carbon dioxide (CO2) incubator at 37°C. The medium was changed every 2 days, and hADSCs from passages 4 to 7 were used in the experiments. 50 µL of serum-free DMEM containing 3 × 105 cells were dispensed over the 3D patch. The hADSCs entered the pockets of the 3D patch after centrifugation at 1,500 rpm for 5 min, and spheroids were formed in the open and open/closed pockets after 24 h.
Table 1
Sample codes and experimental condition of S_3DP.
Sample codes | Pocket type | Patch swelling time | Cell number (cells/patch) | Incubation time |
2D | - | At least 5 min | 3 × 105 | 24 h |
S_OP | Open |
S_OPCL | Open/closed |
Characterization of stem cell spheroid
We observed the spheroid morphology under a light microscope (CKX41, Olympus) after 24 h. Images were captured to confirm the diameter of the spheroids using a light microscope (CKX41, Olympus) and Infinity analyze software (Lumenera Corporation), and the diameter was analyzed using ImageJ software.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted using 1 mL of TRIzol and 200 µL of chloroform. After centrifugation at 11,000 rpm for 10 min at 4°C, 80% (v/v) isopropyl alcohol (in water) and 75% (v/v) ethanol (in water) were used for washing. The samples were then dissolved in RNase-free water. To synthesize complementary DNA, 1 µg of total RNA and the AccuPower® RocketScript™ Cycle RT PreMix were used. The AccuPower® 2X Greenstar™ qPCR MasterMix and QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) were used for qRT-PCR. The relative gene expression was analyzed using the 2^(-ΔΔCT) method and GAPDH served as the internal control.
Table 2
Sequences for primers used in qRT-PCR.
Species | Gene | Primer | Sequence (5’─3’) |
Human | GAPDH | Forward | GTC GGA GTC AAC GGA TTT GG |
Reverse | GGG TGG AAT CAA TTG GAA CAT |
BAX | Forward | GCT ACA GGG TTT CAT CCA GGA TC |
Reverse | CCG TGT CCA CGT CAG CAA TC |
BAK | Forward | CTC AGA GTT CCA GAC CAT GTT G |
Reverse | CAT GCT GGT AGA CGT GTA GGG |
CASPASE-3 | Forward | CCT GGT TAT TAT TCT TGG CGA AA |
Reverse | GCA CAA AGC GAC TGG ATG AA |
CASPASE-9 | Forward | CTC AGA CCA GAG ATT CGC AAA C |
Reverse | GCA TTT CCC CTC AAA CTC TCA A |
BCL-2 | Forward | TCC CTC GCT GCA CAA ATA CTC |
Reverse | ACG ACC CGA TGG CCA TAG A |
BCL-xL | Forward | AAC ATC CCA GCT TCA CAT AAC CCC |
Reverse | GCG ACC CCA GTT TAC TCC ATC C |
VEGF | Forward | GAG GGC AGA ATC ATC ACG AAG T |
Reverse | CAC CAG GGT CTC GAT TGG AT |
FGF-2 | Forward | AGC GGC TGT ACT GCA AAA AC |
Reverse | GTA GCT TGA TGT GAG GGT CG |
HGF | Forward | TCA AAT GCC AGC CTT GGA ATT CC |
Reverse | TCA AGA GTG TAG CAC CAT GGC |
HIF-1α | Forward | CAG TTA CGT TCC TTC GAT CAG TTG |
Reverse | TTT GAG GAC TTG CGC TTT CA |
Western blot analysis
Spheroids were extracted using PRO-PREP™ Protein Extraction Solution to prepare protein samples. Protein concentration was determined using the Bradford assay. Protein samples were boiled at 100°C for 10 min in 4× Laemmli sample buffer containing β-mercaptoethanol, and samples were loaded onto a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. After gel electrophoresis, proteins were transferred from gel to nitrocellulose membrane, and membranes were continuously blocked with 1× TBS-T containing 5% skim milk for 1 h at room temperature (RT). The membranes were incubated overnight at 4°C with primary antibodies: anti-GAPDH (1:3,000), anti-E-cadherin (1:500), anti-BAX (1:250), anti-BCL-2 (1:250), anti-HGF (1:500), and anti-VEGF (1:250). The membranes were washed with 1× TBS-T for 45 min and incubated for 1 h at RT with goat anti-mouse IgG-HRP (1:5,000) and goat anti-rabbit IgG-HRP (1:10,000) secondary antibodies. After incubation, the membranes were washed with 1× TBS-T for 45 min and protein bands were detected using the ECL reagent WESTSAVE UP and an X-ray film. To analyze protein expression, GAPDH and ImageJ software were used as a housekeeping control and an analysis tool.
Angiogenesis antibody array
To profile angiogenesis-related proteins in the conditioned medium (CM) of the spheroids, we used the Proteome Profiler Human Angiogenesis Array. After spheroid formation in the 3D patch, the cells were carefully washed with 1× PBS, and the spheroids in the 3D patch were incubated with fresh serum-free DMEM for 24 h to harvest the CM. The protein concentration of the harvested CM was measured by Bradford assay. The array membranes were blocked according to the manufacturer’s instructions and the buffer was aspirated. The sample/antibody mixtures were added to the membranes and incubated overnight at 4°C. The following day, we washed each membrane with 1× wash buffer and membranes were incubated with streptavidin-HRP for 30 min at RT. After washing, the membranes were covered with Chemi reagent mix and exposed to X-rays. Pixel densities on the developed X-ray film were quantified at each spot using ImageJ software.
Synthesis and characterization of biosealant polymers
To fabricate the biosealant for fixing S_3DP on the heart surface, we synthesized thiolated gelatin (GtnSH) and maleimide-conjugated gelatin (GtnMI) via EDC/NHS-mediated conjugation of thiol and maleimide groups to Gtn backbone. To synthesize GtnSH, we dissolved 250 mg gelatin in 125 mL of deionized water (DIW) at 37°C. After Gtn dissolution, 450.5 mg CYS (2.0 mmol) was dissolved in 2.5 mL DIW and added into the Gtn solution. We homogenously mixed them for 10 min at 37°C. Next, 192.0 mg EDC (1.0 mmol) and 116.0 mg NHS (1.0 mmol) were dissolved in DIW (2.5 mL) in a 10 mL vial. EDC and NHS solutions were sequentially added to Gtn/CYS and allowed to react for 2 h at 500 rpm. After the conjugation reaction, we injected DTT solution (617.0 mg, 4.0 mmol) dissolved in 5 mL DIW into the reactant and incubated for 24 h at 500 rpm. This reactant was serially dialyzed using a dialysis membrane bag (molecular weight cut-off = 3.5 kDa) against a 5 mM HCl solution for 36 h, followed by a 1 mM HCl solution for 24 h. The solution was changed twice to remove unconjugated residual chemicals such as unconjugated CYS, byproducts of the EDC/NHS reaction, and unreacted DTT molecules. To obtain the GtnSH polymer, we froze dialyzed the GtnSH solution at the − 80°C and lyophilized it for 5–6 days. The obtained polymers were stored in a desiccator until use.
To synthesize GtnMI, we dissolved 250 mg Gtn and 211.2 mg MHA (1.0 mmol) in 50 mL and 120 mL DPBS solution at 37°C, respectively. Each solution was mixed for 10 min at 37°C. Then, 230.1 mg EDC (1.2 mmol) and 161.1 mg NHS (1.4 mmol) were dissolved in 5 mL DPBS within a 10 mL vial, respectively. The EDC solution was mixed with the NHS solution and added to the Gtn/MHA mixture. After 2 h of reaction, the reactant was dialyzed using a dialysis membrane bag (molecular weight cut-off = 3.5 kDa) against DIW for 72 h to remove the unconjugated MHA molecules and byproducts of the EDC/NHS reaction. After dialysis, the GtnMI solution was frozen at -80°C and lyophilized for 5–6 days. Lyophilized GtnMI were stored in a deep freezer at -80°C without light.
A proton nuclear magnetic resonance (1H-NMR) spectrometer (Agilent 400–MR, Agilent Technologies, CA) was used to characterize the chemical structures of GtnSH and GtnMI. We prepared 600 µL GtnSH and GtnMI solutions (10 mg/mL) using D2O and analyzed each chemical structure.
Next, Ellman's assay was performed to determine the functional groups of the biosealant polymers. To measure the thiol content of GtnSH, we prepared 0–1 mg/mL L-cysteine solutions using DIW to obtain a standard curve. Next, 100 µL GtnSH solution (1 mg/mL) dissolved in DIW and standard solution were mixed with 100 µL Ellman’s reagent solution. After 20 min of reaction in the dark, the absorbance of the samples was measured at 405 nm using an ultraviolet (UV) spectrophotometry (Multiskan EX, Thermo Scientific, Rockford, IL, USA). Thiol content was calculated from a thiol standard curve at known L-cysteine concentrations (0.001–0.04 mg/mL). To measure the maleimide content of GtnMI, we performed a modified Ellman’s assay by introducing a thiol-ene reaction. We prepared a 0.04 mg/mL L-cysteine solution as a substrate for the thiol-ene reaction. After mixing 50 µL 0.04 mg/mL L-cysteine solution with 50 µL standard MHA solution or 50 µL GtnMI solution, we incubated them to induce the thiol-ene reaction for 10 min without light. We next added 50 µL Ellman’s reagent solution to quantify the degree of thiol group reduction depending on the content of the maleimide group in the standard solution and GtnMI solution. After 20 min of incubation in the dark, we measured the absorbance at 405 nm using UV spectrophotometry (Multiskan EX, Thermo Scientific, Rockford, IL, USA). The maleimide content was calculated from the maleimide standard curve at known MHA concentrations (0.005–0.04 mg/mL).
Fabrication of tissue adhesive biosealant
We fabricated a tissue adhesive biosealant by simply mixing GtnSH, GtnMI, and calcium peroxide (CaO2) solutions (volume ratio of GtnSH: GtnMI: CaO2 = 10:9:1). In this system, CaO2 was used as an additional crosslinker to enhance the mechanical property of the biosealant. We first dissolved the 20 mg GtnSH and GtnMI in 200 µL and 180 µL prewarmed DPBS, respectively. We loaded the GtnSH solution into a 1 mL commercial syringe (Korea Vaccine Co., Ltd., Gyeonggi-do, Korea). Next, a 20 µL CaO2 solution (0–2wt%) prepared using 1M Tri-HCl was mixed with 180 µL GtnMI solution and loaded into another 1 mL commercial syringe. Finally, both syringes were equipped with a dual syringe kit, and injected the solution to fabricate the tissue adhesive biosealant.
Rheological analysis of biosealant
We determined the elastic modulus (G′) of the biosealant using a rheometric fluid spectrometer (DHR-1, TA instruments, New Castle, DE, USA) in oscillatory mode. We prepared each 150 µL GtnSH and GtnMI/CaO2 solution and equipped them into a dual syringe kit. Next, we injected a biosealant solution on the parallel plate (diameter, 20 mm) with a gap of 600 µm and performed dynamic time sweeps on samples depending on CaO2 concentrations at a frequency of 0.1 Hz and strain of 0.1% at 37°C. To prevent solvent evaporation, a solvent trap was filled with DIW.
Tissue adhesive test of biosealant
The tissue adhesiveness of the biosealant was measured using a universal testing machine (UTM, UNITEST M1, TEST ONE, Busan, Korea) with a load cell sensor (LCK1205-K010) according to the modified ASTM standard F2255-05 method. We prepared the decellularized porcine skin (round shape, 10 mm diameter) using a 0.1% SDS solution and a freeze dryer. Next, the decellularized porcine skin was attached to an acrylic plate (1 × 4 cm) using cyanoacrylate. After swelling of decellularized porcine skin, we injected a biosealant solution using a dual syringe filled with 100 µL GtnSH and GtnMI/CaO2 solutions between two pieces of decellularized porcine skin. We stabilized samples for 30 min under a force of 100 g at 37°C within a humid chamber. The tissue adhesion force was measured at a speed of 1 mm/min. To evaluate the feasibility of the tissue adhesive biosealant in vivo, we treated biosealant to various mouse tissues, such as the liver, kidney, heart, spleen, lungs, and skin. Various organs and skin tissues were harvested and rinsed with DPBS to remove blood and body fluids. Next, we cut the tissues in half and treated the tissue surfaces or cut edges with biosealant. We stabilized them for 5 min at 37°C within an incubator and lifted them to confirm the tissue adhesion of the biosealant. The animal study was performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the Incheon National University (INU-ANIM-2022-02).
Cytocompatibility of biosealant
We evaluated the cytocompatibility of polymers and biosealants using HDFs. All reagents and solutions used in this experiment were sterilized by UV irradiation for 15 min or syringe filtration with a pore size of 0.2 µm. To assess the cytotoxicity of GtnSH and GtnMI, the WST-1 assay (Roche) was performed according to the manufacturer’s instructions and our previous reports [61, 62]. Briefly, we seeded 1.0 × 104 cells in a 96-well plate (SPL Life Sciences, Korea) with 200 µL of DMEM-HG supplemented with 10% (v/v) NBCS and 1% (v/v) P/S at 37°C in 5% CO2. We prepared different concentrations (0.5–2.0 mg/mL) of GtnSH and GtnMI media using DMEM-HG. After 24 h of cell seeding, we treated polymer media to HDFs and cultured for 24 h. Next, we treated 10% WST-1 solution to HDFs and incubated for 20 min. The absorbance of the suspension was measured at 450 nm using a microplate reader. The cell viability of the polymers was calculated as a percentage of the control cells (untreated cells, TCPS). To determine the cytotoxicity of the biosealant, we performed a WST-1 assay as described above. In brief, HDFs (1.0 × 104 cells/well) were seeded in a 96-well plate (SPL Life Sciences, Korea) with 100 µL of DMEM-HG supplemented with 10% (v/v) NBCS and 1% (v/v) P/S at 37°C in 5% CO2. Next, we prepared the biosealant eluates by incubating 40 µL biosealant droplets with 193 µL media for 24 h. Next, we treated 100 µL serially diluted eluates to HDFs and incubated for 24 h. Then, HDFs were incubated with 10% WST-1 solution for 20 min, and the absorbance was measured at 450 nm. The cell viability of the biosealant was calculated as a percentage of the control cells (TCPS).
In vivo biodegradability and tissue compatibility of biosealant
To evaluate the biodegradability of the biosealant, we subcutaneously injected the biosealant solution into mice (6-week-old female C57BL/6N mice) using a dual-syringe kit in vivo. All polymers, reagents, and solutions were sterilized by UV irradiation for 15 min or filtered through a syringe filter (pore size: 0.2 µm). After a week of stabilization, the mice were anesthetized with isoflurane in balanced oxygen. Next, we shaved dorsal hair and cleaned their skin with 70% ethanol and povidone. We prepared 200 µL biosealant solution and subcutaneously injected it into mice (6-week-old female C57BL/6N mice) using a dual syringe kit in vivo. For eight weeks, we monitored the change in biosealant volume and harvested the biosealant with the surrounding tissues from the mice. The tissue compatibility of the biosealant was evaluated using subcutaneous implantation and H&E staining. After eight weeks of subcutaneous injection, we harvested various organs and fixed them using 10% formalin. The animal study was performed according to a protocol approved by the IACUC of the Incheon National University (INU-ANIM-2022-02).
Acute MI model of rats and transplantation of S_3DP in MI model in vivo
All animal experiments and surgical procedures were approved by the IACUC of the Samsung Medical Center (SMCIACUC2021-03-23-001). Male SD rats (8–10-week-old, 200–250 g) were anesthetized with isoflurane gas, endotracheally intubated, and ventilated with air using a small animal ventilator (Harvard Apparatus, Hopkinton, MA, USA). After opening the chest, MI was induced by permanent ligation of the left anterior descending coronary artery (LAD) using 6 − 0 polypropylene. Ischemia of the anterior wall of the left ventricle (LV) was confirmed by the myocardium turning red to pale pink, and the animals were randomly divided into five groups (n = 5 per group). After LAD ligation, S_3DP was immobilized on the ischemic heart surface using the biosealant. The biosealant was applied along the outside of the patch and allowed to stabilize for 3 min. After confirming that the biosealant and the 3D patch were fixed to the surface of the heart, the chest cavity was closed using 4 − 0 surgifit sutures. The chest muscles were then closed using 6 − 0 polypropylene, and the skin was sutured using 6 − 0 black silk sutures.
Table 3
Sample codes and compositions.
Sample codes | MI induction | Treatment of biosealant | 3D patch transplantation (Pocket type) | Spheroid |
Normal | X | X | X | X |
MI | O | X | X | X |
SO | O | O | X | X |
PO | O | O | O (Open/closed pocket) | X |
S_OP | O | O | O (Open pocket) | O |
S_OPCL | O | O | O (Open/closed pocket) | O |
Echocardiography
The rats were anesthetized after 28 days of MI and S_3DP transplantation, and echocardiography was performed for cardiac function evaluation using a VisualSonics Vevo 2100 (VisualSonics Inc., Toronto, ON, Canada). All echocardiography measurements were performed by a single-blind investigator. The left ventricular internal dimension at end-diastole (LVIDd) and left ventricular internal dimension at end-systole (LVIDs) were measured using the two-dimensional (2D) targeted M-mode to calculate the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). LVEF and LVFS were calculated using the following equations [7, 9, 31, 35]:
-
LVEF = [(LVIDd3 - LVIDs3)/LVIDd3 ×100%]
-
LVFS = [(LVIDd - LVIDs)/LVIDd ×100%]
Histological analysis
After echocardiography, whole hearts were harvested from the sacrificed rats and fixed with 10% formalin. The hearts were sliced into three sections along the transverse axis below the ligature to the apex at 2 mm intervals. The samples were dehydrated in a series of ethanol, embedded in paraffin, and cut into 4 µm-thick sections. The sections were deparaffinized in xylene, ethanol, and distilled water and stained with H&E and Masson’s trichrome to visualize cardiac fibrosis. Stained slides were scanned using the Aperio ScanScope AT Slide Scanner (Leica Biosystems Inc., Buffalo Grove, Illinois, USA) at × 20 magnification, and all images were captured using ImageScope software (Leica Biosystems Inc.). The infarction size and fibrosis area of the LV were determined using the following formula [40]:
-
Infarction size = [(epicardial circumference of the LV infarcted area + endocardial circumference of the LV infarcted area)/(epicardial circumference of the total LV area + endocardial circumference of the total LV area) × 100%]
-
Fibrosis area = [(fibrosis area of LV/total area of LV) × 100%]
The LV wall thickness was measured in the infarcted and border zones. Each zone was divided into six equal segments, and LV wall thickness was calculated by averaging the thicknesses of the six segments [5]. All histological analyses were quantified using ImageJ software.
Statistical analysis
The GraphPad Prism 5 and 7 software (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. Statistical analysis was performed using a t-test and one-way analysis of variance (ANOVA) with the Bonferroni test, and all quantitative data were presented as mean ± standard deviation (S.D.). Statistical significance was set at p < 0.05.