Synthesis of polycaprolactone-co-poly (ethylene glycol)-co-Polycaprolactone (PCEC) copolymers

The PCEC triblock copolymer was prepared by ring-opening copolymerization of ε-caprolactone (ε-CL, 98%, Aldrich) initiated by polyethylene glycol (PEG, Mn = 1500 Da, Aldrich) using stannous octoate (Sn(Oct)2, 95%, Aldrich) as a catalyst [26]. Before starting the polymerization reaction, ε-CL monomer was distilled under the reduced pressure at 90 °C after being treated with CaH2 for 24 h to remove the inhibitor. Also, PEG was dried in a vacuum oven for 72 h at 70 °C before use, as mentioned previously [27, 28]. The PCEC copolymer was prepared as follow: ε-CL (0.175 mol, 10.0 g) and PEG (0.01 mol, 5.0 g) were added into two necked vessels under a dry nitrogen atmosphere and the reaction system was kept at 120 °C for 1 h. Then, Sn(Oct)2 (0.1% Wt of the total mass of monomers) was added to the reaction vessel and continued to stir for about 6 h under the nitrogen flow at 120 °C with a rotating speed of 350 rpm. After the gel formation and production of the light-yellow polymer, a few droplets of HCl 37% were added to terminate the polymerization reaction and the resultant copolymer was cooled to the room temperature. In the end, the obtained PCL-PEG-PCL triblock copolymer was dissolved in dichloromethane and precipitated in the chilled diethyl ether. The yield of the polymerization was obtained at about 93%. The purified polymer was kept in an air-tight bag before use.

Characterization of PCEC CopolymersFourier Transform Infrared Spectroscopy (FTIR)

The functional groups of PCEC copolymer were assessed by using the computerized ATR-FTIR spectroscopy (Burker Tensor 27, USA). The spectrum of the sample was recorded from 4000 − 400 cm− 1.

1H Nuclear Magnetic Resonance analysis (1H NMR)

The chemical composition and molecular weight of the copolymer were characterized by 1 H-NMR (400 MHz) spectra in CDCl3 solvent by using a Spectrospin Avance spectrometer. Chemical shift for the proton of CDCl3 is reported at 7.26 ppm.

Gel permission chromatography (GPC)

A Shimadzu LC-20 A apparatus along with the refractive index detector was used to determine the weight average molecular weight (Mw) and molecular weight distribution (PDI) of the PCEC triblock copolymers. Tetrahydrofuran (THF) was used as a mobile phase with a flow rate of 1.0 ml/min at 35 °C. The Mw of the copolymer was calculated from the polystyrene standard with narrow molecular weight distribution.

Investigation of phase transition temperature

The sol-gel-sol phase diagram of the thermosensitive copolymer PCEC in water was studied by using the test tube inverting method in a 4 ml screw-capped vial (diameter 1.1 cm) based on the flow (sol) and nonflow (gel) criterion [29]. The solutions with a given concentration were prepared by rapidly dispersing in 55 °C water followed by cooling in an ice bath. The solutions were kept at 4 °C for 12 h before measurements. To determine the phase transition temperature, the samples were measured from 10 to 60 °C. The samples with no visual flow after inverting the vial for 30 s were considered as a gel.

Effect of PCEC thickness on cell sheet formation

To assess the effect of PCEC thickness in the cell-sheet formation, two volumes of the polymer gels (0.5- and 1.0 ml) were added into wells of 6-well format with equal diameters. Then, 1.5 × 106 rBMSCs cells were seeded on top of the polymers with different thicknesses and allowed to produce cell sheets for 21 days. The viability and strength of the cells were checked after sheets were released from each well.

Rat bone marrow-derived mesenchymal stem cells culturing

The experimental procedures were approved by the Animal Ethical Committee of Tabriz University of Medical Sciences with the number of the IR.TBZMED.REC.1399.036. The rBMSCs were isolated according to the previously described protocols [30]. Briefly, about 106 cells of passage 3 were cultured on a T75 tissue culture flask at 37 °C in a humidified atmosphere of 5% CO2 containing DMEM supplemented with 10% fetal bovine serum (GIBCO, Invitrogen). The culture medium was changed every 3 days. When cells reached more than 80% confluency, the cells were detached by using 0.25% trypsin / 1 mM EDTA. The cells were aliquoted into three flasks and considered for the next experiments.

Osteogenic and adipogenic differentiation of rBMSCs

Osteogenic/adipogenic differentiation of rBMSCs was performed according to the previous reports [31]. The rBMSCs were cultured at a density of 5 × 10³cells/cm2 on a 6-well plate with a culture medium supplemented with 10% MSC Qualified FBS (GIBCO, Invitrogen), incubating at 37 oC with 5% CO2. After 24 h, the cells reached about 60–70% confluency, and then the medium was changed with differentiation media (GIBCO, Invitrogen). Osteogenic differentiation media supplemented with 10% FBS, 50 µM dexamethasone, and 100 nM indomethacin and adipogenic differentiation media contained the 0.05 mM vitamin C, 0.1µM dexamethasone, and 10 mM β-glycerophosphate. Control groups were cultured just with 10% FBS in DMEM. The cultures were incubated at 37 °C in 5% CO2 for 21 days and the medium was changed twice a week. At the end of 21st day, the medium was removed and rinsed once with warmed PBS (37 oC). Next, the cells were stained with alizarin red and oil red solution to confirm the osteogenic and adipogenic differentiation, respectively. The images were recorded by using a light microscope.

MTT assay

First of all, rBMSCs were seeded into 96-well plates at a density of about 104 cells per well and then treated with sodium selenite (Se), vitamin C (Vc), sodium selenite + vitamin C (Se + Vc), and Trolox (Tx, 1, 10, 0.1, 0.01, and 0.001 µM). Their cytotoxic effect on the seeded cells was assessed by MTT mitochondrial metabolism evaluation method [14]. After 1, 3, and 6 days, the treatments were aspirated and replaced with MTT solution (5 mg/mL) followed by incubation for 4 h, then, the medium was removed, and 100 µL DMSO was added to dissolve the formazan crystals; finally, glycine buffer was added. Cell viability was measured at 540 nm by using an ELISA plate reader (Multiskan MK3, Thermo Electron Corporation, USA). Cell growth was compared with the control routine media exposed cells. The results were reported as mean ± SD for three independent samples.

Fabrication of rBMSCs sheet

First, the 6 well plates were coated by PCEC copolymer, then the seeding of rBMSCs was performed at a density of about 5 × 106 cells per well by applying the routine medium as the control group, and four other groups including the medium supplemented with Vc (50 µg/ml), Se (0.1 µM), Se + Vc (50 µg/ml + 0.1 µM), and Tx (25 µg/ml). The cells were continuously cultured for 2 weeks without passaging, and each medium was replaced every 3 days, cell adhesion, viability, and proliferation were monitored by a phase-contrast microscope (Olympus) until sheets formed and reached confluence. Next, the cell sheets were harvested by temperature reduction by keeping the wells in an ice bath for 5 min. Cell sheets released from the PCEC sol form were observed with an inverted microscope (Olympus).

Histological analysis of sheets

The harvested cell sheet was rinsed with sterile PBS at 37 ºC, fixed in 4% paraformaldehyde for 1 h at 4 °C in the refrigerator, then, embedded in paraffin and sliced into 5-µm sections. To examine the histology of the prepared cell sheets, hematoxylin and eosin (H&E) staining was performed by common methods, and their photographs were captured by a light microscope (Olympus).

Field Emission Scanning Electron Microscopy (FESEM)

To assess the morphology and other details of the released sheets, they were processed and visualized by the FESEM method. In this way, they were fixed by a PBS solution of 2.5% glutaraldehyde (pH 7.3) at 4 °C and then 2% osmium tetroxide in the same buffer solution for about 1 h. Subsequently, they were subjected to dehydration procedures in several concentrations of the ethanol and t-butyl alcohol (50, 75, 95, and 100%) for 15 min and then, lyophilized overnight led to the cell sheets by osmium coating. Then, the apical and basal surfaces of the sheets were analyzed by the FESEM.

Quantitative real-time PCR (q RT-PCR)

The expression level of the stemness genes (Sox2, Oct-4, Nanog), selenium-dependent enzymes (TRX, GPX-1), and aging regulator gene (Sirt1) were measured by the real-time PCR in each released cell sheet. The total RNA was extracted using Trizol reagent (Thermo scientific) according to the manufacturer’s protocols. The cDNA was synthesized by using a Thermo scientific kit (Revert Aid cDNA synthesis kit) and q RT-PCR was done by SYBR green Master Mix (Thermoscientific). The cDNAs, primer, and master mix cocktail were incubated for 3 min at 95 ºC, started loop 40 cycles at 94 ºC for 30 s, 60 ºC for 40 s, 72 ºC for 45 s, closed loop, and finally at 72 ºC for 5 min. The results were reported as fold changes that were calculated by the 2-ΔΔCT method. The sequences of the primers are shown in Table 1.

Table 1 The sequences of primers in q RT-PCR. Statistical analysis

Statistical analyses were done by using the GraphPad Prism version 8 (GraphPad Software, Inc., La Jolla, CA). All experiments were performed in triplicated and reported as mean ± SD. The two statistical methods, ANOVA or Student’s T-Test were performed for comparing the groups. The significance level was defined as P-values < 0.05.