GelMA bioink preparation
Cardiomyoblasts (H9C2 cells) obtained from Procell (Wuhan, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 IU/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a 5% CO2 atmosphere. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), GelMA powder, and the UV light source (405 nm) were purchased from the SuZhou Intelligent Manufacturing Research Institute (SuZhou, China). LAP was dissolved in phosphate-buffered saline (PBS) to a concentration of 0.25% (w/v) and heated at 55 °C for 15 min to ensure complete dissolution. GelMA powder was dissolved in LAP solution at a concentration of 6% at 70 °C for 30 min and filtered through a filter membrane (0.22 µm, Millipore, Billerica, MA, USA) before use. Cardiomyoblasts were collected from the culture dish and dispersed into the GelMA solution (37 °C) to form a single-cell suspension at a cell density of approximately 5 × 106 cells/mL. The cell-laden GelMA solution was then loaded into a 5-mL syringe equipped with a 25 G needle, which was fixed at the extrusion printhead.
3D-bioprinting
Scaffolds were fabricated using a customized extrusion-based 3D printer BioMaker (SUNP BIOTECH, China). Computer-aided design software was used to produce a G-code file for printing. A 3D scaffold model with dimensions of 10 mm width, 10 mm length, and 1 mm height was designed with a 1-mm filament grid and 0.2-mm layer thickness. Before bioprinting, the cell-laden GelMA bio-ink was placed at 4 °C for approximately 30 s until it changed from liquid to semi-solid, and then placed in the 3D printer for bio-printing. The cell-laden GelMA bio-ink was extruded onto a moving platform based on the model described above, and Biomarker Software Suite 1.0.7 (SUNP BIOTECH, China) was applied to monitor and manage the printing process. The 3D GelMA scaffolds containing H9C2 cells were printed into a Petri dish and immersed in DMEM at 37 °C with 5% CO2 after UV light exposure (wavelength: 405 nm) for 30 s to photo-crosslink the bio-inks to ensure their long-term stability. All printing processes were performed at room temperature with the nozzle set at a moving speed of 10 mm/s.
Optimization of LIPUS treatment protocol and dosage
The LIPUS exposure device (Chongqing Haifu Medical Technology Co., Ltd., Chongqing, China) comprises an array of five transducers (34.8 mm in diameter), which is specifically drafted for a 6-well culture plate. A 6-well plate was deposited on the transducers with a thin layer of ultrasound coupling agent between them. The parameters of the LIPUS device were an intensity of 1.0 W/cm2, duty cycle of 20%, and pulse repetition frequency of 1 kHz; four frequencies (0.5, 1.0, 1.5, and 2.0 MHz) were used. Scaffolds in the LIPUS group were exposed to LIPUS, whereas those in the control group received sham irradiation (LIPUS was turned off).
Cytocompatibility tests
A Cell Counting Kit-8 (CCK-8; Servicebio, Wuhan, China) assay was performed to measure the proliferation of H9C2 cells. After 1, 4, and 7 days of treatment with LIPUS or sham irradiation, scaffolds with H9C2 cells were incubated in CCK-8 solution for 4 h. Next, 10 µL of the supernatant was transferred into a 96-well culture plate, and its absorbance at 450 nm was measured using a microplate reader (EnSight; Perkin Elmer, Waltham, MA, USA). To reduce the effect of non-clay adsorbing the staining solution, scaffolds without seeded cells were used as the blank group.
Cell viability was evaluated using the Live and Dead Cell Double Staining Kit (EFL-CLD-001, SuZhou) after treatment with LIPUS or sham irradiation for 1, 4, and 7 days. The scaffolds were washed twice with PBS, and then immersed in live/dead staining solution of 10 mL PBS containing 5 µL calcein-AM and 5 µL propidium iodide (PI) solution according to the manufacturer’s instructions. After incubation at 37 °C for 45 min, the staining solution was removed, and the scaffolds were washed with PBS. Fluorescence staining was analyzed using confocal laser scanning microscopy (ECLIPSE TI; Nikon, Tokyo, Japan). Cell viability was calculated from the number of green (representing live cells) and red points (representing dead cells).
Flow cytometry
For the cell cycle assay, after LIPUS or sham irradiation treatment, cell-laden GelMA scaffolds were cut into small pieces and lysed using GelMA Lysis Buffer (EFL-GM-LS-001, SuZhou, China), harvested, and fixed with pre-cooled 75% ethanol at 4 °C overnight. After centrifugation, the cells were incubated with PI and RNase A at 37 °C for 30 min in the dark. The cell cycle stages were analyzed at an excitation wavelength of 488 nm using flow cytometry.
Apoptosis of H9C2 cells was determined using an FITC Annexin V and PI double staining kit (Servicebio, Wuhan, China). After different treatments, the cells were collected, resuspended, and incubated with 5 µL of Annexin V and 5 µL of PI. After incubation for 20 min in the dark at room temperature, the cells were stained for 15 min, and a flow cytometer (BD Biosciences, San Jose, USA) was used to detect apoptosis. The data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
Western blot analysis
H9C2s were pre-treated with U0126 (20 µmol/L) (specific inhibitors of the ERK1/2 pathway) or LY294002 (50 µmol/L) (specific inhibitors of the PI3K-Akt pathway) for 1 h before 3D bioprinting. After bioprinting, the scaffolds were treated with LIPUS or sham irradiation and then lysed with GelMA Lysis Buffer for 2 h. The H9C2 cells were isolated by centrifugation and lysed in RIPA lysis buffer, after which the proteins were obtained using centrifugation. Protein concentrations were determined using the Bradford protein assay kit. The protein samples were separated using SDS-PAGE and then electro-transferred onto polyvinylidene fluoride membranes (Millipore). After washing, the membranes were blocked with 5% skim milk for 1 h, and then incubated overnight at 4 °C with specific primary antibodies for Bcl2, Bax, Caspase-3 (all from ASPEN, Wuhan, China), ERK1/2, phospho-ERK1/2, PI3K, phospho-PI3K, Akt, and phospho-Akt (all from ASPEN). After washing three times, the membranes were incubated for 1 h at room temperature with secondary antibodies (ASPEN). The BeyoECL Plus kit was used for color development according to the manufacturer’s instructions (Beyotime, Shanghai, China).
Statistical analysis
At least three independent experiments were performed for all tests. All data were analyzed using SPSS 25.0 (SPSS, Inc., Chicago, IL, USA) and expressed as the mean ± standard deviation. Mann-Whitney U test or Kruskal-Wallis test were used to analyze continuous variables across groups. For multiple comparisons, two-sided P values were adjusted to control the false-discovery rate using the adaptive Benjamini-Hochberg procedure.