α-Minimum Essential Medium (α-MEM), Hank's balanced salt solution (HBSS), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA, USA). TGF-β1 was purchased from Peprotech (Cranbury, NJ, USA). Smooth muscle growth medium-2 (SmGMTM-2) was purchased from Lonza (Bazel, Switzerland). Polycaprolactone (PCL; molecular weight (Mw) = 45,000 Da), calcium chloride (CaCl2), and polyethylene oxide (PEO; Mw = 900,000) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium alginate with 0.42 mannuronic acid to guluronic acid (M/G) ratio (LF10/60; FMC Biopolymer, Drammen, Norway) was kindly provided by Pharmaline (Suwon, South Korea). 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Merck (Darmstadt, Germany). The antibodies used in this study are listed in Table S1.
Isolation of MSCs from human palatine tonsil tissues and cell culture
After obtaining informed consent from patients, human palatine tonsil tissues were isolated from patients undergoing tonsillectomy due to chronic tonsillar hypertrophy and/or chronic tonsillitis in the Department of Otorhinolaryngology-Head and Neck Surgery, Pusan National University Hospital [31, 32]. To isolate tonsil-derived MSCs, tonsil tissues were washed with phosphate-buffered saline (PBS) and digested at 37°C for 30 min with 0.075% type I collagenase. The enzyme activity was neutralized with α-MEM supplemented with 10% FBS and 10% penicillin-streptomycin. The dissociated cells were filtered through a sterile 70 µm cell strainer and cultured in 5% CO2 at 37°C. When the cell confluence reached 70–80%, cells were washed twice with HBSS, treated with 0.05% trypsin-EDTA, and incubated in 5% CO2 at 37°C for 3–5 min. The suspended cells were harvested in medium containing FBS and centrifuged at 500 × g for 4 min. MSCs were sub-cultured at a split ratio of 1:3 or 1:4, and cells at passage 7–9 were used for experiments. To differentiate MSCs into SMCs, MSCs were serum-starved in α-MEM basal media for 24 h, treated with 2 ng/mL TGF-β1 for 4 days, and maintained in SmGMTM-2 medium.
To extract total protein, cells were treated with lysis buffer [Tris-HCl (20 mM), EGTA (1 mM), EDTA (1 mM), NaCl (10 mM), phenylmethylsulfonyl fluoride (0.1 mM), Na3VO4 (1 mM), sodium pyrophosphate (30 mM), β-glycerol phosphate (25 mM), and 1% Triton X-100, pH 7.4], followed by sonication and centrifugation at 12,000 rpm at 4°C for 10 min. Lysates were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk buffer for 2 h and incubated overnight with primary antibodies. For visualization with chemiluminescence, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, and the signal was detected with the ECL kit (Cytiva, Mariborough, MA). The membrane was washed four times for 20 min with 1× Tris-Tween buffered saline.
Cell growth on two-dimensional (2D) surface with/without aligned fibers
A rectangular glass (10 × 10 mm) was used as the non-fibrous surface. Then, aligned alginate fibers were deposited by electrospinning onto the glass under a 10.5 kV high-voltage power source, 140 mm nozzle-to-electrode (NtE) distance, 0.25 mL/h flow rate, and 3 min electrospun time. The fibers were aligned using rectangular copper electrodes (75 × 25 mm) and a metal nozzle (180 µm). Then, MSCs and SMCs at 62500 cells/droplet, which is mathematically equivalent to the number of cells laden on the CE scaffold, were seeded on the glass with or without aligned fibers.
Preparation of cell-laden bioink
To obtain a cell-laden bioink, 2 wt % alginate and 3 wt % PEO (A2P3) were dissolved in triple-distilled water and stirred for 2 days at 4°C. Then, either MSCs or SMCs at 1 × 107 cells/mL were added to the A2P3 solution.
Fabrication of cell-printed and cell-electrospun scaffold using MSCs and SMCs
A 3D printer (DTR3-2210-T-SG, DASA Robot, Bucheon, South Korea) was used to fabricate a PCL strut (300 µm in diameter and 30 mm in length). Pneumatic pressure (340 kPa) was applied within the heated metal barrel containing PCL at 100°C, and the melted PCL was extruded through a metal nozzle (350 µm inner diameter (ID)) at 10 mm/s. CP was subsequently performed on printed PCL struts. The cell-laden bioink was printed through a nozzle (ID: 180 µm) using a pneumatic pressure of 120 kPa and a nozzle moving speed of 10 mm/s.
For CE, a high-voltage power source (SHV300RD-50K, Convertech, Gwangmyeong, South Korea) and a syringe pump (KDS 230, NanoNC, Inc., Seoul, South Korea) were prepared. The cell-laden bioink was supplied at 0.25 mL/h, and electrospun under a 10.5 kV high voltage direct current (HVDC) and 140 mm NtE distance. The electrospun fibers were deposited on the PCL strut, which was fixed between parallel cylindrical electrodes at a distance of 30 mm.
To fabricate a PCL fibrous mat, electrospinning was performed using 10% PCL dissolved in methylene chloride and dimethylformamide at a 4:1 ratio. The PCL fibers were collected on a grounded rotating drum at 1500 rpm using 0.20 mL/h flow rate and 0.1 kV/mm electric field (12 kV HVDC and 120 mm NtE distance) for 4 h.
Characterization of the surface topography of scaffolds
To observe the surface topography of the scaffolds, optical images were captured using a BX FM-32 optical microscope (Olympus, Japan). To obtain microscale images, the samples were placed on double-sided carbon tape and sputter-coated with gold, and their images were captured using a scanning electron microscope (SEM; SNE-3000M; SEC Inc., Suwon, South Korea). Stress-strain curves were obtained using a microtensile tester (Toptech 2000; Chemilab, Suwon, South Korea) under uniaxial stretching at 0.2 mm/s.
Analysis of cell viability and differentiation on cell-laden scaffolds
Cell viability at various cell culture periods (in situ, 7, 14, and 21 days) was observed using a Live/Dead Viability/Cytotoxicity Kit (Invitrogen). The samples were immersed in a solution containing 0.15 mM calcein AM and 2 mM ethidium homodimer-1 for 30 min, and images were captured using light microscopy. Cell viability was calculated as the ratio of the number of live cells to the total cell number using Fiji software.
Cell nuclei and F-actin were visualized with DAPI (blue) and phalloidin (green) staining, respectively. The samples were fixed in formaldehyde solution (3.7% in Tris-buffered saline [TBS]) for 12 h at 4 oC and treated with 0.3% Triton X-100 in TBS for 10 min at 25 oC. Then, the samples were treated for 1 h at 37 oC with a DAPI (5 ×10− 6 M; Invitrogen)/phalloidin (15 U/mL; Invitrogen) staining solution. Fluorescence images were captured using a confocal microscope (LSM 700; Carl Zeiss, Germany) and analyzed using Fiji software.
Differentiation of MSCs and SMCs was observed using immunofluorescence staining. Samples were fixed and permeabilized prior to DAPI/phalloidin staining. The samples were then immersed in 1% bovine serum albumin (BSA) in TBS for 1 h at room temperature. Primary antibodies against α-SMA, fibronectin, calponin, collagen I, and collagen IV at 1:200 in TBS were added to the samples and incubated overnight at 4 oC. Subsequently, the samples were stained with secondary antibodies conjugated with Alexa Fluor 488 or 594 (Invitrogen). Images were captured using confocal microscopy and analyzed using Fiji software.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
The expression levels of connexin 43 (Cx43), smooth muscle protein 22 alpha (SM22α), desmin, and smoothelin (SMTN) were analyzed using RT-qPCR. Briefly, total RNA was isolated using TRI reagent (Sigma-Aldrich) according to the manufacturer’s instructions. The purity and concentration of the isolated RNA were determined using a spectrophotometer (FLX800T; Biotek, VT, USA). cDNA synthesis was performed using 500 ng RNase-free DNase-treated total RNA using a reverse transcription system (FSQ-201; Toyobo, Osaka, Japan). The gene expression level was measured by the comparative Ct method using the StepOne Plus RT-PCR system (Applied Biosystems, Foster City, CA, USA). GAPDH gene expression was used as an internal control. The primers used in this study are listed in Table S2.
Repair of esophageal wounds using SMC-laden patch
Six-month-old Sprague-Dawley rats (Central Lab. Animal Inc., Seoul, Korea), weighing approximately 680–700 g, were used in this study. Rats were anesthetized with isoflurane (Baxter International Inc., USA). An anesthetic delivery equipment was used to administer a gas mixture of isoflurane and O2 via inhalation into the rat’s respiratory system. Before anesthesia, isoflurane was evaporated in a vaporizer (Harvard Apparatus, USA), and the concentration was set to 5% and adjusted to 2% during the operation. The gas flow rate of O2 was maintained at 0.5–1 LPM.
To create an esophageal defect model, the center of the ventral neck was shaved, an incision of approximately 2 cm made, and the esophagus located in the tracheoesophageal structure. To equalize the size of the esophageal defect, a biopsy punch (Miltex, KAI Industries, Japan) of 2 mm diameter was used to completely puncture the muscle and mucosa layer of the esophagus. The patches were cut with a biopsy punch of 4 mm diameter and placed over the esophageal defect, and the punctured section of the esophagus was sutured with a 9 − 0 nylon suture (AILEE Co., Korea). The incised skin was sutured using a 7 − 0 nylon suture. All surgical procedures were performed using sterile instruments and disinfection procedures. Fasting was abstained for 3 days after surgery, sterilized water was fed on the 4th day, and food was served on the 6th day. Two weeks after the surgery, the rats were sacrificed by CO2 euthanasia. All experiments were reviewed and approved by the Institutional Review Board of Pusan National University Hospital (PNUH-2021-188).
For histological analysis of esophageal regeneration, esophageal tissues were excised with tracheal tissue to support the esophagus. For routine staining, the tissues were fixed with cooled acetone. To obtain cryosections, the tissue was perfused with sucrose solution, embedded in optimal cutting temperature compound, and frozen at -80°C. For routine staining, the tissue sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome stain. Stained sections were scanned using an Axio Scan.Z1 (Carl Zeiss Microscopy, Germany).
For immunohistochemical analysis, the tissue samples were fixed in 4% paraformaldehyde overnight and embedded in paraffin. To detect smooth muscle regeneration and vascularization, esophageal tissue sections were stained with anti-SM22α, anti-IL-B4, anti-vimentin, and anti-desmin antibodies. Macrophages were stained using anti-CD68 antibody. Alexa Fluor 568 goat anti-mouse and Alexa Fluor 488 goat anti-mouse antibodies and Alexa Fluor 488 streptavidin were used to label the fluorescence, and DAPI was used to stain the nuclei. The stained sections were visualized under a laser confocal microscope (Olympus FluoView FV1000) and Axio Scan.Z1 (Carl Zeiss Microscopy). The fluorescence levels were quantified in a high-power field using ImageJ software.
The data are presented as the mean ± standard deviation and were analyzed using SPSS 18 software (SPSS, Inc., Chicago, IL, USA). Differences among multiple groups were compared using analysis of variance, and the statistical significance was represented as p* < 0.05, p** < 0.01, and p*** < 0.001.