Isolation of adipose-derived MSCs and bone marrow-derived MSCs
following the protocol described previously [22], adipose tissues were isolated from the abdominal region of male C57 / BL6 mice and digested with 1 mg/ml collagenase type 1 (Sigma Aldrich) for 1 hour in a shaker incubator at 37 °C. Cells were isolated by centrifugation and Red blood cells were removed from the sample by RBC Lysis buffer (Qiagen, Hilden, Germany). In the end, the cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) and Incubated at 37 ° C and 5% CO2. The mixture cultures were passaged for purification and homogenization of the cell population. The bone marrow's from Tibia was isolated from a male C57/BL6 mice. First, the bones were cut and bone marrow [22] was extracted into a petri dish using a syringe with a 22-gauge needle containing DMEM using the flushing method. After washing, the centrifugation was done. The resulting cell pellet was suspended in DMEM and incubated at 37°C with 5% CO2. Further passaging was carried out to ensure cell purification..
Characterization of mouse adipose tissue-derived and bone marrow-derived MSCs
After two passages, flow cytometry was used to describe the adipose tissue-derived and bone marrow-derived MSCs. At first, the cells were treated with trypsin 0.25% and EDTA 0.01% for 5 minutes. Then, it was washed 3 times with phosphate buffer and allowed to react with FITC conjugated anti-CD90, PE anti-human CD 105 (mesenchymal markers), FITC-conjugated anti-CD45 (hematopoietic marker) all from Santa Cruz. Isotype antibodies were utilized as controls for each cell population. The ability of the cells to differentiate into adipocytes and osteocytes was assessed following established protocols [23].
Briefly, mesenchymal stem cells were cultured in a differentiation medium for 3 weeks in accordance with the protocol. The differentiation to adipocytes was investigated by staining Oil Red (Sigma-Aldrich) while differentiating into osteocytes was evaluated with Alizarin Red (Sigma-Aldrich) staining. Then, The stained cells were examined by inverted phase-contrast microscope (Nikon, Tokyo, Japan).
Synthesis of alginate hydrogel scaffold
A certain amount of alginate powder (biochemical grade, molecular weight = 398, Sigma Aldrich, USA) was dissolved in 0.9% NaCl solution (Sigma Aldrich) which resulted in solution of 1.2 mg sodium alginate. Then, it was mixed with 50 mL CaCl2 solution for 30 minutes and washed twice with PBS. In the end, it was covered with PBS at 12 well plates then left at room temperature.
MSCs encapsulation in an alginate hydrogel scaffold
To transfer the cells to the alginate scaffold, 1 ml of alginate solution was added to cell deposition containing 106 cells at the third passage. After 15 minutes, alginate bubbles were converted into hydrogels, and the remaining calcium chloride was removed. It was then washed with a Tris buffer (Angus chemical company/ USA) and then once with the culture medium. After washing with the medium, the F12 medium (Invitrogen) containing 10% Fetal Bovine Serum (FBS, Sigma Aldrich, USA), 1% penicillin/streptomycin (pen/strep, Invitrogen, USA) were added and finally, the plates were transferred to the incubator.
Evaluation of cell proliferation by MTT assay
In this study, MTT assay was used to evaluate the growth and proliferation of mesenchymal stem cells in alginate scaffolds. After transplantation of mesenchymal stem cells into scaffolds during days 1, 3 and 7, the scaffold propagation power was compared to control. To perform this test, the MTT solution at a concentration of 0.05 mg/ml was poured onto the scaffolds and the plates were completely covered with foil and placed in the 37 °C incubator. After 4 hours, the changes were examined with Invert microscope. After the formation of purple crystals, they were dissolved with Dimethyl Sulfoxide solution (DMSO, Merck) and the absorbance of the solution was calculated with ELISA reader at 570nm wavelength. Wells without a scaffold was also used as control that the cells were directly cultivated on them.
In vivo study of BMSCs and Ad MScs encapsulated in alginate hydrogel scaffold treatment on burn infections
The burn model was made using adult male mice 6-8 weeks and weighing 25-35 g. Mice were kept in individual cages based on ethical protocols that accepted by IUMS ethical committee (IR.IUMS.rec1395.29887). To maintain their physiological rhythms, a 12-hour light and 12-hour dark cycle was implemented. The temperature of the storage room was adjusted to 22.0 ± 2 °C. The mice were anesthetized with ketamine and xylose and after shaving the dorsal area, the burn was induced by an aluminum piece of 1.5×1.5cm2 (Fig. 1). The burned area was inoculated with 5 × 108 Colony – Forming Units (CFU) MRSA. Treatment was performed 24 hours after infection in mice with MRSA. A total of 7 groups were established as outlined in Table 1. In the end, the mice were followed for 7 days.
Counting MRSA at the site of the burn wound
At first, the skin was homogenized in 1 ml of phosphate buffered saline (PBS) by a homogenizer and 9 ml of PBS was added, in result, dilutions of 10-1 to 10-12 were prepared. They were then cultured in Nutrient agar medium (Merck) and incubated for 24 hours at 37 °C and CFU was determined for each group.
Histopathological analyses of burn wound biopsies
Skin biopsy specimens from each group were fixed in 10% formalin (Sigma-Aldrich) to preserve their structure. The specimens were placed in paraffin and cut by the microtome. Then, H & E staining was used to observe the epidermis.
Immunohistochemistry (IHC) was used to evaluate collagen type 1. First of all, the samples were washed with PBS and placed in 2N HCl for 30 minutes, then it was added to the borate buffer and washed after 5 minutes. After that, the primary antibody collagen I (mouse monoclonal AB; Abcam, 1:100) was added to the sample and left to incubate overnight. The following day, the sections were washed to remove any excess primary antibody. Next, the secondary antibody (Rabbit polyclonal IgG; Abcam, USA, 1:200) was added to the sample and incubated for 37 minutes at 90 °C. After washing, 4, 6-diamidino-2-phenylindole (DAPI, Sigma) was added to samples and quickly removed. In the end, the markers were examined using a fluorescence microscope (Olympus BX51, Japan) with a 400 lens to visualize the markers.
Statistical analysis
The obtained data were expressed as mean of standard deviation. Statistical analysis was performed by one way ANOVA. P < 0.05 was considered significant.