Materials

Sodium alginate was obtained from CARLO ERBA Reagent (France). Chitosan (medium molecular weight) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Sigma-Aldrich Co. α-Tocopherol and ascorbic acid were purchased from Sigma (USA). Dulbecco’s modified Eagle's low-glucose medium (DMEM) was purchased from GIBCO (Life Technologies). Trypsin and penicillin–streptomycin (Pen/Strep) were obtained from Bio idea, and fetal bovine serum (FBS) was purchased from AnaCell. Acetic acid (glacial) 100% was purchased from Merck.

Preparation of Chi/Alg/ AA/TP hydrogel

The chitosan/alginate (CA) hydrogels containing CA microparticles and bioactive agents (ascorbic acid and α-tocopherol) were prepared by ionic gelation according to the following steps: 1% glacial acetic acid to dissolve chitosan at 20–25 °C overnight used. Alginate was dissolved in deionized water at 20–25 °C overnight. Then, equal proportions of alginate and chitosan were blended together at room temperature for 25 min and a 1:1 chitosan-alginate solution (1% chitosan and 1% alginate) was obtained. Afterward, 400 IU of α-Tocopherol was added to a 10 ml chitosan and alginate mixture to prepare the drug-containing hydrogel. Once again, 100 μM ascorbic acid was added to the chitosan and alginate mixture. Finally, 400 IU α-Tocopherol and 100 μM ascorbic acid were added to 10 ml chitosan and alginate and vigorously stirred to form a uniform solution and microparticles. All prepared hydrogels were immersed in 1% w/v CaCl2 solution in deionized water for 10 min to form cross-linking. Hydrogels were then immersed in PBS (pH = 7.4) for 10 min to remove unbound cross-linkers. The samples were then lyophilized in a freeze dryer for 48 h at − 50 °C. In the present study, four groups of hydrogels, including chitosan/alginate (CA), chitosan/alginate/ascorbic acid (CA/AA), chitosan/alginate/α-tocopherol (CA/TP), and chitosan/alginate/ascorbic acid/α-tocopherol (CA/AA/TP), were prepared.

Hydrogel characterizationMorphological properties

To investigate the surface morphology and porosity of composite hydrogels and the CA microparticles incorporated in them, the freeze-dried samples, including CA, CA/AA, CA/TP, and CA/AA/TP, were analyzed by scanning electron microscope (MIRA3 FEG-SEM, Tescan, Brno, Czech Republic) at an accelerating voltage of 30 kV and resolution of 1 nm. To this end, the samples were cut into 1cm × 1cm dimensions, coated with gold, and then evaluated with an SEM microscope.

Fourier transform infrared spectroscopic analysis (FTIR)

Infrared spectroscopy was used for the chemical characterization of composite hydrogels, the molecular composition of AA and TP, and chemical bonds. The samples were prepared as compressed KBr disks at room temperature and placed in a magnetic holder. Finally, FTIR analysis of freeze-dried samples, including pure alginate and chitosan, CA, CA/AA/TP, as well as a powder form of TP and AA, was done using a Thermo Scientific Nicolet iS5 FTIR spectrometer in the range between 4000 and 400 cm−1.

Wettability measurement

The surface hydrophilicity of composite hydrogels was investigated by measuring the contact angle. A contact Angle Meter (IFT-CA, CA-ES20, Iran) was used to investigate freeze-dried hydrogel’s wettability. In this way, the samples were cut into dimensions of approximately 2 × 3 cm2, distilled water was dripped on the surface, and the contact angle of the water formed at the interface between the water and the sample surface was recorded.

Weight loss analysis

To determine in vitro degradation rate, freeze-dried hydrogels (including CA, CA/AA, CA/TP, CA/AA/TP) were cut and weighed. The solid samples were immersed in phosphate-buffered saline (PBS) with pH 7.4 and placed in a 37 °C incubator. Then, at intervals of 1, 2, 3, 7, 14, and 21 days, they were removed from the buffer and dried at 37°C, and then their dry weight was measured. To measure the degree of degradation, the obtained weights were placed in the following formula (1) [2]:

$$}\,\,}\,\left( \% \right)\, = \,\frac} \times 100$$

(1)

where W0 represents the initial dry weight of the hydrogel composite and W1 is the final dry weight.

Swelling behavior

To evaluate the fluid uptake of hydrogels, including CA, CA/AA, CA/TP, and CA/AA/TP samples, freeze-dried samples were cut, their dry weight was recorded, and then they were immersed in PBS buffer (pH = 7.4) and were placed in a 37 °C incubator. After predetermined times (2, 4, 24, 48, and 72 h), the samples were removed from the buffer, and after removing the excess water from the swollen hydrogels using filter paper, they were quickly weighed. The swelling capacity percentage was obtained using Eq. (2) [2]:

$$}\, }\, \left( \% \right)\, = \,\frac} \times 100$$

(2)

where W2 represents the swollen weight of the composite hydrogels and W1 is the dry weight.

Release study

UV–visible spectroscopy was used to investigate the release of α-TP and AA from composite hydrogels in vitro. CA hydrogel (15 mg) containing 9 mg TP, as well as CA hydrogel containing 100 μM AA, was placed separately in a falcon tube containing 20 ml PBS buffer (pH 7.4) and incubated in a shaker incubator (50 rpm) under temperature 37 °C (optimal concentration of drugs was determined based on articles [2, 23]). At specified time intervals (6, 12, 24, 48, 72, and 120 h), the buffer containing the released drug was changed with 20 ml of fresh buffer. The absorbance intensity of the buffer containing the released drug for TP and AA was recorded at λmax = 290 nm and λmax = 265 nm, respectively, by UV–Vis spectrophotometer. Equation 3 was used to determine the release rate of TP and AA [2].

$$} \,\,} \,\left( \% \right)\, = \,\frac}\,\, }\,\,}\,\, }\,\,}\,\,}}}}\,\,}\,\,}\,\,}\,\,}\,\,}\,\,} }} \times 100$$

(3)

Hemolysis study

To investigate the blood compatibility of freeze-dried hydrogels, including CA, CA/AA, CA/TP, and CA/AA/TP, first, all samples were cut into 3 mg pieces and placed in 2-ml microtubes. Then, 200 µl of fresh rat blood were collected in citrated tubes, and 800 µl of normal saline solution was added to the samples. For the positive control group, 800 µl of deionized water was added to 200 µl of blood, and for the negative control group, 800 µl of normal saline was added to 200 µl of blood. After incubation at 37 °C for 1 h, the samples were centrifuged for 10 min at 3000 rpm, and then the absorbance of the supernatant was obtained at λmax = 540 nm using a UV–vis spectrophotometer. The percentage of hemolysis was obtained with Eq. (4) [24]:

$$}\,\left( \% \right)\, = \,\frac}}} - OD_}\,\,}}} }}}\,\,}}} - OD_}\,\,}}} }} \times 100$$

(4)

Cell culture experimentsIsolation of rMSCs

Rat bone marrow mesenchymal stem cells (rMSCs) were isolated from the femur and tibia of male Wistar rats (12 weeks old). The animals were maintained in accordance with the guideline set forth by the local Animal Care Committee (IR.TBZMED.VCR.REC.1400.034) in the Animal House of Tabriz University of Medical Sciences. To isolate bone marrow mesenchymal stem cells (BM-SCs), the rats were first euthanized with high doses of ketamine and xylazine, femur and tibia bones were harvested, and the epiphysis was aseptically cut off. Then, bone marrow was flushed into the cell culture flasks by Low-Glucose Dulbecco’s Modified Eagle’s Medium (DMEM-LG; Gibco, Germany), supplemented with 10% (v/v) fetal bovine serum (FBS; Anacell, Iran) and 1% pen-strep (Biosera, Sussex, UK). The flasks were placed in an incubator at 37 °C in a humidified atmosphere of 95% air and 5% CO2. After 24 h, the supernatant was aspirated, and adherent cells were washed twice with pH 7.4 PBS. Finally, cells were grown in a standard growth medium until reaching 80% confluence.

Characterization of rMSCs by flowcytometry

To characterize rMSCs, in the third passage, the cells were trypsinized, and after washing with PBS, ten tubes were selected, and the cells were separately incubated with anti-rat monoclonal antibodies containing anti-CD34, -CD45, -CD73, and -CD105 for 30 min at 4 °C. Then, for washing, 500 µl of the PBS solution was added to the tubes and centrifuged for 5 min at a speed of 1500 rpm. After removing the supernatant, 250 µl of PBS was added to the cells, and analysis was performed using BD FACS Calibur (BD biosciences, San Jose, CA, USA).

Cell viability studies

To evaluate biocompatibility, hydrogels were first cut and sterilized with 70% v/v alcohol. Then, they were washed three times with PBS and transferred to 96-well plates. Next, rMSCs were cultured on the hydrogels at a density of 1 × 104 cells in DMEM supplemented with 10% (v/v) FBS, 1% of pen-strep and incubated in a humidified incubator (37 °C, 5% CO2). After 72 h, the supernatant was removed from the 96-well plates, and 0.2 ml MTT (0.5 mg/ml) was added to each well and incubated for four hours in an incubator at 37 °C. MTT was then replaced with 0.1 ml DMSO for 30 min. Finally, absorbance was measured by an ELISA reader at 570 nm to determine cell viability.

H&E staining

Hematoxylin and eosin (H&E) staining was used to show the presence of cells inside the hydrogels. In this regard, rMSCs were cultured on the CA hydrogels. After 3, 5, and 7 days, cell-containing hydrogels were fixed by 10% formalin solution, and after washing with PBS, the samples were immersed in Xylene, 100% ethanol, 95% ethanol, 80% ethanol, and deionized H2O, respectively. Finally, the samples were stained with hematoxylin and eosin and examined using light microscope images.

DAPI staining

DAPI staining was used to stain the nuclei of living cells in hydrogels. In brief, CA hydrogels were placed in 6-well plates and sterilized with 70% v/v alcohol. After washing with PBS, rMSCs were cultured onto the hydrogels, and after 24 and 72 h, the culture medium was removed. Then, cell-containing hydrogels were washed with PBS and incubated with 3.7% formaldehyde at room temperature for 10 min. Next, the cells were washed 1–3 times with PBS. After removing the buffer, 2 mL DAPI staining solution was added to each well and incubated at room temperature in the dark to bind to AT-rich DNA. After 15 min, the stain solution was removed, and the stained samples were washed with PBS. Finally, cell-containing hydrogels were imaged by an Olympus BX50 fluorescence microscope.

Hypoxia induction and real-time PCR

MSCs were cultured onto the hydrogels in separate groups for three days. Then, to apply hypoxia, the cells were exposed to a medium containing 100 µl of CoCl2 for 8 h. Then, the supernatant of all groups was removed, and Trizol reagent (MXcell-Tehran-Iran) was used for RNA extraction. For the cDNA synthesis, 1ng of RNA was removed from each group and subjected to cDNA synthesis according to the cDNA synthesis kit protocol (Yekta Tajhiz Azma Iran). Real-time PCR was used for looking at gene expression of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor-A (VEGF-A), and transforming growth factor- β1 (TGF-β1) using the gene-specific primers in Table 1. β-actin was considered a housekeeping gene. Samples include N-CA in normoxic condition (N) as a control group and H-CA, H-CA/AA, H-CA/TP, and H-CA/AA/Tp in hypoxic condition (H) to monitor the expression of VEGF-A and TGF-β1. Also, N-CA and H-CA were used to monitor the expression of HIF-1α. Method 2(-Delta Delta C (T)) was used to analyze relative changes in gene expression in real-time PCR experiments [25].

Table 1 Primer sequences used for real-time quantitative PCR (q-PCR)Statistical analysis

The mean ± standard deviation of each sample group represents the results. GraphPad Prism software (version 8.3.0, USA) was used to perform statistical analysis based on a two-way ANOVA and Tukey post hoc analysis for all tests except the real-time PCR assay. T-test and one-way ANOVA analysis were used for real-time PCR assay. The experiments were performed in triplicate.