5.1. Human bone marrow mesenchymal stem cell culture
The hMSCs used for the experiments were kindly provided by the Stem Cell Technology Research Center (Iran) with the ethical approval certificate. The hMSCs were cultured at a density of about 5× 103 cells/cm2 in DMEM high glucose medium, 10% FBS, and penicillin/streptomycin in an incubator at 37°C and 5% CO2. The cell culture medium was replaced every 48 hours. The hMSCs harvested at almost 80% confluence of about 2× 104 cells/cm2 will result in hMSCs doubling per passage. Following passage 3, they were characterized to be MSCs by observing spindle-shaped cells, plastic adherence feature, and owning the capability of differentiation to osteogenic cells (the data not shown).
5.2. Osteogenic differentiation of hMSCs
For differentiation of MSCs, a differentiation medium has β-glycerol phosphate (1.8 g per 5 ml of deionized water), dexamethasone (0.0017 g per 50 ml culture medium), and ascorbic acid (0.19 g per 20 ml of culture medium) prepared. Following treatment with differentiation medium, cells maintained at 37°C and 5% CO2 for two weeks in an incubator, and every 48 hours medium was changed.
5.3. MTT assay
Three concentrations (100-200-300 IU/mL) of Nisin were evaluated for the optimal Nisin dosage used for the preconditioning of MSCs on the 1st, 3rd, and 5th days of culture. In each well of a 48-well plate 1×104, MSCs were cultured and incubated in 37°C and 5% CO2. After the 1st, 3rd, and 5th days of culture, 200 µl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well. Then the plate was incubated for 3 hours. In the next step, 100 µl of Dimethyl sulfoxide (DMSO) solution was added and the solution of each well was transferred to a 96-well plate. Then the readings were performed at 540–630 nm by an ELISA reader.
5.4. Acridine Orange Staining
Acridine Orange staining was performed to investigate the growth and proliferation of MSCs on the 1st, 3rd, and 5th days of culture. 200–300 µl of Acridine orange solution was added to each well and incubated for 5 minutes at 37°C with 10% CO2. In the next step, the cells were washed twice with PBS and photographed by the fluorescence microscope.
5.5. Alkaline Phosphatase Activity
The activity of alkaline phosphatase as a marker in differentiated bone marrow cells was investigated on the 7th and 14th days. At first, 200 µl of Ripa solution was added to each well and the pipette was completely taken and the solution of each well with scaffolds was transferred to 1.5 ml vials and vortexed. The vials were centrifuged (4°C, 15,000 rpm, 15 min). Using an alkaline phosphatase kit (Pars Azmoon, Tehran, Iran) and adding 150 µl of R1 and R2 solutions, plus 50 µl of samples, which transferred to a 96-well plate, readings at 450 nm were carried out by the ELISA reader.
5.6. Calcium Content Assay
The amount of calcium sediment accumulation in differentiated MSCs to osteocytes was evaluated on days 7th and 14th of the differentiation. 200 µl of the hydrochloric acid solution was added to each well of a 6-well plate. After pipetting, the contents of each well were transferred to 1.5 ml vials and shaken for 40 minutes. Using a Calcium Detection Kit (Pars Azmoon, Tehran, Iran) and adding 150 µl of R1 and R2 solutions plus 50 µl of samples, they were transferred to a 96-well plate and read out at 570 nm by the ELISA reader.
5.7. Alizarin Red Staining
To assess the quality of calcium deposition, on the day 14th of differentiation was performed. The wells were removed and washed once with PBS and twice with deionized water. 4% Paraformaldehyde was added to each well and placed at 4°C for 20 minutes. In the next step, and Alizarin solution was poured onto cells and scaffolds. Then it was washed with PBS for 5 minutes and photographed under a reverse microscope.
5.8. Imaging by scanning electron microscopy
To investigate the morphology, and adhesion as well as biocompatibility of MSCs with PLLA scaffolds, we used scanning electron microscopy.
After 14 days, MSCs were differentiated to the osteocytes all of the wells were washed with PBS. 2.5% Glutaraldehyde was added to each well and washed twice with PBS for 30 minutes. Then, alcohol dehydration was carried out (60-100-100%) each well for 10 minutes. The fibers were coated with gold when they dried. Then they were scanned by a scanning electron microscope.
5.9. Synthesis of Poly-L-lactic acid (PLLA) nanofibers
Nanoparticles of poly-lactic acid were made using the electrospinning method. 0.43 gr of polymeric poly-lactic acid was dissolved in 5 mL of chloroform solution. Then the solution was transferred to the syringe of the device. It was subjected to a voltage of 15 kV with a speed of 0.4 mL/h. Then, the fibers are made on the aluminum plate.
5.10. Immunocytochemistry
To evaluate the Osteocalcin and Osteopontin proteins, on the 14th day of differentiation, the supernatant was removed and cells were washed with PBS. To fix the samples were added, 4% paraformaldehyde, 20 minutes, 0.4% Triton X100, 10 minutes, respectively. Then, the goat’s serum of 5%, the primary antibodies of Osteocalcin (1:100), and Osteopontin (1:200) were added. After 30 minutes’ incubation (37°C, 5% CO2) were washed three times with Phosphate Buffered Saline (PBS). In the next step, the secondary antibody, Fluorescein isothiocyanate (FITC) (1:100), was added and incubated at room temperature for 1 hour. Then, it washed again with PBS. Cell cores were stained with a DAPI (4′,6-diamidino-2-phenylindole) color of 0.1 µg/mL and photographed with a fluorescence microscope.
5.11. Analysis of metastasis genes expression by quantitative RT-PCR analysis
5.11.1. RNA extraction
To determine the expression of alkaline phosphatase, Osteocalcin, Osteonectin, collagen type 1, and RUNX-2 on days 7th and 14th, all tested specimens were firstly subjected to RNA isolation. The supernatant of the wells was discarded and washed with PBS. 700 µl of RNA X plus solution was added to each well and placed in an incubator for 10 minutes. The contents of the wells were transferred to a microwave oven, and 200 µl of cold chloroform was added to each vial, and centrifugation (12000 rpm, 15 min, at 7°C) was performed. Then 100 µl of supernatant was transferred to a new vial and then added 400 µl of cold isopropanol. The supernatant was discarded Again, and 1 ml of 75% alcohol was added to each vial. Then centrifugation (7800 rounds, 10 minutes, low temperature) was performed. After drying completely, 25 µl of diethyl pyrocarbonate (DEPC) Water was added to each vial and Vortexed. The quality of RNAs was measured by spectrophotometry (NanoDrop, Wilmington, DE). The concentration of RNA was read at 280/260 nm of optical absorption. The proper absorption is between 1.8-2.0, indicating the proper concentration of RNA in the specimens.
5.11.2. cDNA synthesis
Appropriate concentrations of RNA were picked up, with about 2 µl of each primer, 1 µl oligo-dT, and 1 µl random hexamer primer. The obtained solutions were placed in the PCR apparatus at 65°C for 5 minutes. Next, 2 µl including RTase Buffer 10X and O-1MDTT, 1 µl Reverse Transcriptase, and RNase Inhibitor were added, so the final volume reached 20 µl. The new times and temperatures were set for 5 minutes at 25°C, 60 minutes at 55°C, and 4 minutes at 80°C, for 35 cycles.
5.11.3. Real-time PCR
After cDNA synthesis, they were subjected to real-time PCR protocol according to the kit manufacturer's instructions. It is performed using DNA primers and adding SYBER-green qPCR Master Mix. Then the vials are put in a Rotor-gene (Corbett) real‐time analyzer. The times and temperature settings included 20 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C, for 38 cycles.
5.12. Statistical analysis
All experiments were repeated three times. The results were reported as mean ± standard deviation. Statistical analysis was performed using SPSS software by t-test. P ≤ 0.05 was considered for significant changes. Real-time PCR data were analyzed using Rotor-Gene Q and REST software.
The data that support the findings of this study are available on request from the corresponding author.