2.1. Materials
Sodium alginate (SA) with a number-average molecular weight of 32,000 and polydispersity index of 1.8, and the M/G ratio of 0.82 (details in supplementary information), polycaprolactone (PCL) with a molecular weight of 80,000 g mol−1, 3-[4,5-dimethylthianzol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) compounds were purchased from Sigma-Aldrich. Sodium bisulfite and sodium nitrite as the sulfating agents were purchased from Merck, Germany. Merck or Sigma-Aldrich supplied all other chemicals.
2.2. General procedure for preparation of surface-modified electrospun PCL scaffolds
2.2.1. Synthesis of sodium sulfated alginate (SSA)
In brief, sodium sulfated alginate was synthesized through one-step substitution reaction of sodium alginate and the sulfating agent solution. For this aim, sodium alginate (5 g) was added to the aqueous solution of sodium bisulfite (11.10 g) and sodium nitrite (1.75) to obtain a solution with final solid content of 9 wt.%, and the reaction was performed under vigorous stirring at 90 °C for 1.5 h. After that, the solution was dialyzed using a dialysis bag (MWCO, 3500) against distilled water for 48 h and lyophilized to obtain the purified powder of sodium sulfated alginate36.
2.2.2. Preparation of electrospun solutions and their electrospinning
First, 0.6 g PCL was dissolved in an acetic acid/formic acid solvent system with a volume ratio of 1:9 to obtain the solution with solid content of 5%, and stirred 2 h before use. For the electrospinning process, a 5-mL syringe with a 19-gauge needle was filled with the polymer solution. The values of flow rate and applied voltage were 1 mL h-1 and 25 kV, respectively. Also, the tip-to-collector distance, and collector rates were 120 mm and 250 rpm, respectively. The electrospinning process (Co881007 NYI, ANSTCO, Iran) was carried out using a horizontal system with a cylindrical collector covered by aluminum foil at room temperature.
2.2.3. Surface activation of nanofibrous scaffolds using cold atmospheric plasma
To improve the hydrophilic properties of electrospun nanofiber surfaces and creation of active functional groups for subsequent interactions, the surface of the nanofibrous mat was activated using helium cold atmospheric plasma (HCAP). Plasma was applied to the nanofibrous mat for 3 min at a pressure of 1 mbar.
2.2.4. Surface modification of nanofibrous mat using sulfated alginate
HCAP activated scaffolds were cut at 0.5×0.5 cm2 sheets. After that, they were washed with distilled water and placed in sulfated alginate aqueous solutions with different concentrations of 3, 5 and 7 mg mL-1 for 24 h. The surface-modified scaffolds were placed in distilled water for another 24 h and dried in vacuo.
2.3. Measurements
2.3.1. Physicochemical characterization of electrospun scaffolds
The chemical structure of neat and sulfated alginate was studied using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) analysis (RX I Spectrum, PerkinElmer, USA) equipped by ATR accessories. The infrared spectra of the samples were measured with 32 scans at the 8 cm-1 resolution and over a wavelength range of 4000-400 cm-1. The proton nuclear magnetic resonance (1H NMR) spectroscopy was performed to further characterize the chemical structure of sulfated alginate using Bruker DRX-500 Avance spectrometer (Germany). The spectra were recorded in D2O as the solvent.
The degree of sulfation (DS), the average number of sulfate groups per uronic acid residue, was measured using elemental analysis method (Costech 4010, Italy). The experimental DS was calculated using the following equation:
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)
Where [S] is sulfur content (%) of sulfated alginate. The molecular weight (MW) of sodium sulfated alginate was measured using GPC 1100 Agilent equipped with PL gel column and water as the eluent.
For investigation of PCL nanofibers, morphology and the impact of surface modification using HCAP and sulfated alginate on nanofibrous structure, the scanning electron microscopy (SEM) (Model Vega, Tescan Co., Czech Republic) was employed. ImageJ software was used for measuring the fiber diameters. Besides, the ATR-FTIR spectra of neat and surface-modified nanofibrous mats were used to confirm successful surface modifications of mats.
2.4. Evaluation of the biological activities
2.4.1. Culture of mesenchymal stem cells (MSCs)
The MSCs of bone marrow were obtained from Stem Cell Technology Research Center. The cells were cultured in a Dulbecco’s modified eagles medium (DMEM, Bioidea, Iran) containing 10 wt.% of fetal bovine serum (FBS, Gibco, Germany) in an incubator with a CO2 injection capability of 5% and a humidity of 95% at 37 °C.
2.4.2. Cell seeding and culture on nanofibrous scaffolds
After sterilizing the scaffolds by UV-rays for 20 min, they were incubated in culture medium overnight before cell seeding in order to make sure for removal of surface contaminants and facilitate protein adsorption and cell attachment onto the scaffold surface. The MSCs were seeded on the scaffolds at the density of 1Í104 cells cm-2 in culture medium supplemented with 10% FBS up to 72 h. Moreover, the cells tendency to the scaffold was measured by reverse microscope. The cellular culture was maintained in an incubator at 37 °C with 5% CO2.
2.4.3. Cell morphology on scaffolds
Furthermore, the morphology of seeded MSCs on the surface-modified PCL (SM-PCL) nanofibrous mat was examined using SEM. After washing the cells that were seeded (1Í104 cells/well) on scaffolds with PBS, the attached cells were fixed by paraformaldehyde solution (2.5% v/v). To dehydrate the cells, scaffolds were placed in a series of EtOH with concentrations from 60% to 100%. Then, SEM images were obtained at accelerating voltage of 2 kV after gold sputter coating.
2.4.4. In vitro cytocompatibility evaluation of nanofibrous scaffolds
To evaluate the MSCs viability and proliferation on nanofibrous scaffolds, the MTT assay was used. Briefly, MSCs were seeded at the density of 1Í104 cells cm-2 scaffolds. At three time points of 24, 48 and 72 h, the samples were transferred into new wells, and the MTT solution was added to each well, after which the plates were incubated in the dark at 37 °C for 3 h. The absorbance of the solution was measured at 490 nm. The color absorbance was measured with an ELISA Reader at a wavelength of 570 nm (BioTek EL Í800). The experiments were run in triplicate.
2.4.5. Chondrogenic differentiation of MSCs on the surface of scaffolds
The MSCs at a density of 1Í104 cells cm-2 were cultured in 6-well plates from each of two groups with chondrogenic differentiation media (+S) and without chondrogenic differentiation media (-S) (three replicates) up to 21 days. After 24 h of cultivation, the differentiation medium was added to the wells (dexamethasone (1×10-7 µM), ascorbic acid (0.1 M) and Insulin Transferrin Selenium (ITS 1%), all prepared from Sigma Aldrich). After that, on determined time points, the peripheral medium of each well was removed, and the 10% MTT solution was added and incubated for 3 h. Then, the sediments were dissolved in DMSO. Ultimately, the absorbance of the solution was calculated with ELISA reader instrument at a wavelength of 570 nm.
2.4.6. Alcian blue staining of differentiated MSCs
To confirm in vitro differentiation of MSCs towards chondrocytes on SM-PCL nanofibrous scaffolds, the alcian blue staining was performed. For this aim, the MSCs at a density of 1Í104 cells cm-2 were cultured on scaffolds in the presence of a chondrogenic differentiation medium. After predetermined time points of adding the differentiation medium, i.e., 24 h, 7, 14 and 21 days, the scaffolds were fixed by paraformaldehyde, at room temperature for 20 min, then washed with PBS and located in the vicinity of alcian blue (10-15 μL) for 30 min. After that, scaffolds were washed with aqueous HCl (0.1 mol), and observed on the lamella by an inverted microscope.
2.4.7. Reverse transcription polymerase chain reaction (RT-PCR)
In order to extract RNA from differentiated cells on the scaffold, the MSCs at a density of 1Í105 cells cm-2 were cultured in each well of 6-well plates for 21 days in two groups comprising with and without differentiation medium. The total RNA was extracted using the Aria Tous Extraction Kit according to the manufacturer's protocol (Arya Tous, Iran). The first step of RT-PCR relies on primer extension conversion of RNA to complementary DNA (cDNA) by RT enzyme. Then, the polymerase chain reaction (PCR) is performed on samples. Synthesis kit of Arya Tous was used to synthesize cDNA. All primers including type 2 collagen, AGGTCACAGGTTATCCAG R, AGGTCACAGGTTATCCAG β2M F, TGCTGTCTCCATGTTTGATGTATCT R, and TCTCTGCTCCCCACCTCTAAGT were ordered to Takapu-Zist after being designed. All stages were performed to determine the expression of β2M gene and collagen type II with their primers. Finally, they were placed in thermocycler, and the temperature protocol of the kit was conducted for 2 min at 95 °C, 30 s at 94 ° C, 30 s at the specified temperature for the device based on the melting temperature (Tm) of desired genes, and further 30 s at 72 °C to perform the RT-PCR test. The PCR products were taken on a 1.5 wt.% agarose gel, and the gel was stained with SYBR green and photographed with a photo document device.
2.4.8. Immunocytochemistry of differentiated cells
Immunocytochemistry test was performed to confirm the differentiation of MSCs towards cartilage cells. The MSCs at a density of 1Í104 cells cm-2 were cultured for 21 days on the scaffolds in two groups comprising with and without differentiation medium in a 6-well plate. At the designated time, samples were examined in terms of type II collagen (Col II) protein expression. At first, the samples were washed with PBS and placed in paraformaldehyde in a cool place for 20 min. Then, they have rewashed with PBS and incubated first with type II collagen antibody (Santa Cruz Biotechnology, USA) at 4 °C. In order to block the secondary antibody (conjugated with phycoerythrin, Chemicon Temecula, USA) response, the goat serum (10 wt.%) was added for 30 min to the background as an additional color. After constant washing, DAPI compound was added to the samples and immediately removed. After that, they were charged onto the PBS solution and kept at 4 °C. Finally, the samples were observed with an Olympus fluorescent microscopy (Nikon, Japan) to confirm desired markers.
2.4.9. Statistical analysis
The data were statistically analyzed using One-Way ANOVA test to evaluate the statistical significance. The results were analyzed by SPSS 18 software, and the variance was in the significant level of p ≤ 0.05.