All materials were prepared from Sigma Company (Sigma-Aldrich, St. Louis, MO, USA) unless stated otherwise. Ethical approval was obtained from the Institutional ethical review board (approval number; IR. KMU. REC. 1394. 639) at Kerman University of Medical Sciences, Kerman, Iran. Human adipose–derived stem cells (hADSCs) were isolated from three 45 ± 3 old years women after a written consent had been obtained, using a standard cell culture protocol as previously described (article in review). Briefly, for primary culture of hADSCs, the adipose tissues were sliced into approximately 4 mm pieces and after washing with phosphate buffer saline (PBS) containing antibiotics, they were incubated with 0.5 mg/ml collagenase type IV, then centrifuged and the supernatant was removed and the cell pellet was suspended in culture medium containing; DMEM/F12 supplemented with 5% (v/v) FBS penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin-B (2.5 µg/ml) and cultured on plate at 37 °C and 5% CO2. After 24 h, the medium was changed with fresh medium. The medium was replaced every 3 days until the cells reached an approximately 80% confluence.
Phenotypic characteristics by flow cytometry
1 × 105 cells/ml were fixed and incubated for 15 min at 4 °C with a 1:9 dilution of normal goat serum in PBS. The cells were then labeled with the following antibodies: FITC-conjugated anti-CD44, FITC-conjugated anti-CD34 (Chemicon; Temecula, CA, USA), FITC-conjugated anti-CD45 (Ediscience; USA), PE-conjugated-anti-CD73 (BD; San Jose, CA, USA), PE-conjugated anti-CD90 (Dako, Glostrup, Denmark) and PE-conjugated anti-CD105 (R&D; Minneapolis, MN, USA) for 1 h. The cells were washed and analyzed using a FACS Calibur (BD, NJ) machine and WinMDI software (BD Biosciences).
Induction of adipogenic, osteogenic and chondrogenic differentiation
To assess adipogenic differentiation, 1 × 103 cells/cm2 were cultured in adipogenic differentiation medium (DMEM/F12 supplemented with 10% FBS, 100 nM dexamethasone, and 200 nM indomethacin). After 21 days, as accumulation of lipid-rich vacuoles is detected within cells, Oil Red O staining was done. Osteogenic differentiation was induced in osteogenic differentiation medium (50 µg/ml ascorbic acid, 10 nM dexamethasone, and 10 mM -glycerophosphate) with 3–5 × 103 cells/cm2. After 21 days, Alizarin Red S staining, as determination of calcium accumulation assessment, was performed. Chondrogenic differentiation was persuaded by chondrogenic differentiation medium (Invitrogen, USA) in density of 3–5 × 103 cells/cm2. After 21 days, chondrogenesis was evaluated using Alcian blue staining. The slides were fixed with 4% paraformaldehyde for 30 min and washed with PBS.
Fabrication of fibrin scaffold and cell encapsulation
For preparation of fibrinogen solution, fresh frozen plasma (FFP) was obtained from Iranian Blood Transfusion Organization. FFP was thawed in water bath at 37 °C and then 10 ml of it was mixed with 10 ml of Protamine sulfate. The obtained solution was centrifuged at 1800g for 10 min. The supernatant was collected and 0.1 M Sodium citrate was added to the tube. Then, the fibrinogen solution was filtered by a 0.2 µm filter and prepared for usage as cell culture. Mean final fibrinogen concentration was 60 mg/ml. For encapsulation of the hADSCs into the fibrin gel, 200 µL of fibrinogen solution was added to a 5 cc syringe containing 1 × 105 cells in 50 µl CaCl2 (50 mM) and 50 µl thrombin solution (Sigma-T6884, Thrombin from human plasma) (40 IU/ml), and allowed to polymerize for 10 min at 37 °C, CO2 incubator. Thus, hADSCs can be embedded in 3D fibrin gels. When the polymerization has ended and solution became gel, it was carefully immersed in a single well of a 6-well plates containing 2000 µl DMEM/F12 supplemented with 10% FBS, 1% antibiotic solution and 200 µl tranexamic acid, as an anti-fibrinolytic agent, then incubated at 37 °C in a humidified atmosphere containing 5% CO2 for different time points of growth after initial seeding.
Scanning electron microscopy (SEM)
The scaffold was washed with PBS and fixed with 2.5% glutaraldehyde at room temperature. After rinsing the scaffold with PBS and then with double deionized water (DDI), the fibrin scaffold was immersed in liquid nitrogen and kept in a freezer dryer (Super Modulyo, Edwards) . The samples were coated with gold for 180 s by a sputter coater (SC7620, Emitech, UK) and observed under Scanning Electron Microscope (SEM, AIS2100, Seron technology, South Korea) at an accelerating voltage of 20 kV. The diameter of the fibers was calculated from SEM images by image analysis software (Image J, NIH, USA).
Cell cycle analysis
Cell cycle analysis was performed by PI staining. In the first step, the cells from the fibrin scaffold and plate cell culture were detached at the 7th day after initial seeding. Then 1 × 106 cells were put in a 1.7 ml tube and a solution containing 0.25 g sodium citrate, 0.005 g ribonuclease A, 0.025 g PI, and 0.75 ml Triton X-100 was added in 250 ml distilled water and incubated at 4 °C for 30 min. The cells were evaluated by FACS, and percentage of the cells in each phase of the cell cycle (G0/G1, S, and G2/M phases) was calculated using the FlowJo software, version 7.5 (Tree Star, Inc., San Carlos, CA).
Induction of cardiac differentiation
In 2D group, 1 × 104 cells/cm2 were seeded in cardiogenic medium, containing DMEM/F12 supplemented with 100 IU/ml penicillin, 50 µg/ml streptomycin, 5 µl of 0.25 mg/ml amphotericin and 10 µM TSA was added to the cells. In 3D group, the fibrin scaffold was incubated at standard conditions in 6-well plate for 7 days and then 2000 µl cardiogenic medium and 200 µl tranexamic acid were added into a single well and incubated in incubator at 37 °C in a humidified atmosphere containing 5% CO2. For both groups after 72 h, the medium was replaced by DMEM/F12 supplemented with 10% FBS and kept for 1, 2, 3, 4 weeks. To check for cell growth and morphological changes in 2D group, they were observed daily by inverted microscope, but the opacity of the scaffold did not allow the viewing of encapsulated cell in 3D by this type of microscope.
Analysis of cardiogenic gene expressions by qRT-PCR
At weeks 1, 2, 3 and 4 of induction total RNA was extracted from treated and untreated hADSCs samples in 2D and 3D groups. RNA extraction was performed by precipitation method (RIBO-Prep, ILS) according to the manufacturer’s Instructions. After checking RNA integrity by denaturing agarose gel electrophoresis and ethidium bromide staining, cDNA was synthesized by Revert Aid (Thermo Scientific, USA). Quantitative PCR reactions were performed in duplicate on each sample of cDNA.
Aforementioned reactions were accomplished Maxima SYBR Green qPCR Master Mix (Fermentase, USA) in the presence of forward and reverse primers. SYBR green PCR amplifications were initiated at 95 °C for 10 min followed by 35 cycles of 95 °C, 15 s for denaturation; and 60 °C, 30 s for Annealing/extension. We aimed to analyze quantitative PCR on each sample by Rotor Gene 6000 machine (Qiagen, UK) for NKX2.5, Cx43, cTnI, GATA4, HAND1, HAND2, βMHC and MLC2v genes. Levels of each target gene expression were normalized to the expression of GAPDH (as an internal control) and calculated by the ΔΔCt method.
Analysis of NKX2.5 and cTnI by immunocytochemistry
In the 2D group, the induced cells were fixed with 4% paraformaldehyde while in the 3D group, the scaffolds were fixed in 10% neutral buffered formalin for 12 h at room temperature and paraffin block and then sectioned (thickness 5 µm). Then, the fixed cells in 2 groups were incubated with 0.5% Triton X-100 containing PBS and 10% goat serum for 45 min at room temperature. The protocol was followed by a final incubation with NKX2.5 (1:100; Biorbyte, UK) and cTnI (1:100; Abcam, USA) at 4 °C for 24 h. The next day, the cells were washed and incubated with goat anti-mouse IgG (ab150113, Abcam) and goat anti-rabbit IgG (ab150077, Abcam) at room temperature for 45 min. Then, the cell nucleus was stained by 5 µg/ml of Hoescht 33258 for 10 min. The images were taken under a fluorescence-inverted microscope equipped with a digital camera (DP71, Olympus, Japan).
Data from the different tests are presented as mean ± SD of measurements in triplicate and were analyzed by T test and one-way ANOVA followed by post hoc Tukey’s test, after normality assumption. P < 0.05 was considered to be statistically significant.