Preparation of Shilajit
Shilajit was collected from Lorestan province altitudes, in western of Iran. 50 gr powdered Shilajit was dissolved in 500 mL distilled water. This mixture was boiled on high flame to melt, then the flame was declined until it becomes concentrated and black. Shilajit was stored at room temperature and protected from moisturizing. To prepare the appropriated concentrations, the Shilajit extract was dissolved in media or alginate(Alg) and then was sterilized through 0.22 µm syringe filter (Jet Biofil, China).
Inductively coupled plasma mass spectrometry (ICP-MS)
The mineral contents of Shilajit were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). First the sample was crushed into fine powder, then 0.1 gr were treated with concentrated HNO3 and allowed to stand for 30 minutes in room temperature. 500 µL of concentrated HClO4 was added and heated at 100°C. During the heating process, 200-300 µL of concentrated hydrofluoric acid(HF) was added gradually until a clear solution was obtained. This solution was finally used for analysis via ICP spectrometer (ELAN 9000, Perkin-Elmer SCIEX).
Determination of antioxidant capacity
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) method is based on the spectrophotometric measurement of DPPH° concentration at maximum absorption wavelength at 517 nm. The antioxidant molecules can quench DPPH free radicals (reduce the absorption) and convert them to a colorless or bleached product [27].
The DPPH radical-scavenging activity of Shilajit was determined according to the method of Brand-Williams with some modifications [28]. Fifty μl of various concentrations of Shilajit )10, 5,2.5,1.25, 0.625 mg/mL) (dissolved in deionized water) was added to 100 μl methanol containing 0.05 mg/mL of DPPH radical in 96 well-plate. The samples were incubated for 20 minutes at room temperature in dark, and then absorbance was measured with spectrophotometer at 517 nm wavelength.
The experiment was carried out triplicate. Radical scavenging activity was calculated using the following formula:
%Inhibition=[(Ab-Ae)/Ab] ×100
where Ab is the absorbance of the blank sample and Ae is the absorbance of Shilajit
Proton Nuclear Magnetic Resonance (H-NMR)
Shilajit sample was vortexed for about 5 minutes in D2O at room temperature to completely dissolve and the sample was then filtered to remove any particle. The H-NMR spectra of Shilajit was recorded on a Bruker Ascend 300 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a 5 mm probe. Briefly, the spectra were referenced relative to the solvent residual peak (4.7 ppm) with 1024 number of scans, 2 number of dummy scans, 1-second relaxation delay, 24670-time domain, 2.05 acquisition time, and the other parameters were set as default. Assignments of different protons were done based on the chemical shift corresponding to solvent peak (lock frequency) as well as the integrated intensity (proportional to the number of hydrogen) and coupling constant (Spin-Spin Splitting).
Cell isolation and culture
Adipose tissues were collected from the two healthy female patients aged 30 years underwent liposuction surgery and transferred to Cancer and Stem Cell Laboratory of Shiraz Institute for Cancer Research (ICR), Shiraz University of Medical Sciences. The tissues were washed with sterile 1x phosphate buffer saline (PBS) containing 1% of penicillin/streptomycin (P/S, Bioidea, Iran) to remove contaminating blood cells. Then they were cut into small pieces and incubated in 0.2% collagenase type I (Gibco, USA) for 30 minutes at 37 °C and 5% CO2. After digestion, an equal volume of Dulbecco's modified eagle medium (DMEM, Bioidea, Iran) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco, USA) and 1% P/S was added to the samples to neutralize the collagenase type I activity.
Then the obtained soup was centrifuged and cell pellet containing ASCs were resuspended in DMEM supplemented with 20% FBS and 1% P/S. The medium was changed every 3 days and the cells were passaged using 0.25% trypsin solution (Bioidea, Iran). ASCs from passage 3 were used for subsequent experiments.
Characterization of ASCs: Morphology and phenotype
Cell morphology was demonstrated in cell cultures from passage 3 by crystal violet staining (Acros Organics, USA). Cells were stained with 0.5% crystal violet in methanol for 10 minutes at room temperature. Excess stain was rinsed with distilled water and spindle shaped cells were observed with the microscope.
Flow cytometry was performed to analyze the surface antigen expression of isolated ASCs. Briefly, ASCs were stained with fluorescein-5-isothiocyanate (FITC)-conjugated CD45, phycoerythrin (PE)-conjugated CD34 and CD105, and peridinin–chlorophyll–protein (PerCP)-conjugated CD73 antibodies (BD, Biosciences, USA) and were incubated at 4 °C for 15 minutes in the dark. Finally, the frequency of the positive cells for each CD marker was determined using FACS Calibur flow cytometry system (BD, Biosciences, USA). Isotype antibodies were used to exclude non-specific staining of the cells and the data were analyzed by flowjo 7.6 software package (Ashland, San Diego CA, USA).
MTT assay
3-(4,5 dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide) (MTT, Merck, Germany) was used to determine the appropriate and non-toxic dose of Shilajit. In short, 1×104 ASCs/well in 100µL DMEM containing 10% of FBS and 1% of P/S on 96-well plates were incubated with a range of concentrations of Shilajit from 5000 to 9.7 µg/ml. After 24, 48 and 72 hours, the medium was discarded, and 150 μl of 0.1% MTT solution in culture medium was added, and incubated at 37 °C for 4 hrs. Afterward, MTT was removed, and 150 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA) was added and incubated at room temperature for 1h to dissolve the formazan crystal. Finally, the optical density (OD) of the formazan solution was measured with spectrophotometer at 492 nm wavelength.
Scaffold preparation
Alginate(Alg) solution was prepared from low viscosity Alg (Sigma-Aldrich, USA) dissolved in PBS 1x and stirred in a glass beaker overnight at room temperature (25°C) at a final concentration of 1% w/v. For the cell assays, obtained solution was sterile filtered using 0.22 μm syringe filter and stored at 4 °C for further usage.
Differentiation of ASCs
The osteogenic differentiation potential of ASCs was compared in different groups in the presence or absence of Shilajit in 2D and 3D cell culture.
For 3D cultures, 3 × 105 ASCs/mL were suspended in 1% (w/v) Alg with or without 500µg/ml Shilajit, and gelation was induced by adding 2.5% CaCl2. The cells were cultured with or without osteogenic medium (OM, Kiazist, Iran) supplementation for 7, 14 and 21 days. The 3D cultures were divided into four groups: (1) Shilajit/Alg/ASCs+ OM, (2) Alg/ASCs+OM (positive control), (3) Shilajit/Alg/ASCs, and (4) Alg/ASCs (negative control). The cells in negative control cultures received basic medium (DMEM containing 10% FBS and 1% P/S).
For 2D cultures, 3 × 104 cells/mL were seeded in basic medium. Upon 70% confluency, a matched 2D cultures also was performed for 7, 14 and 21 days. The 2D treatments include with the cultures exposed to either OM and 500µg/ml Shilajit at a ratio of 1/2, the same concentration of Shilajit in DMEM, positive control that received OM, or negative control. The media were changed every 3 days for both 2D and 3D groups.
Mineralization assay
After 21 days of differentiation, morphological characterization of encapsulated ASCs within the hydrogels as well as calcium and phosphorus content were respectively evaluated using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). Briefly, hydrogels were lyophilized, samples were fractured into pieces, then, the cut off were coated by gold replica using Q150R-ES sputter coater (Quorum Technologies, London, UK) and SEM imaging were taken using a VEGA3 microscope (TESCAN, Brno, Czech Republic) at 10 kV accelerating voltage. EDX was also performed to evaluate the amount of calcium and phosphorous within the scaffolds. For EDX, three repeats from every group were made.
To assess alkaline phosphatase (ALP) activity, total protein of the differentiated cells was extracted at 7, 14, and 21 days, and ALP colorimetric assay kit (Pars Aazmoon, Iran) was used to evaluate the ALP activity in lysates. To do this, at each point of time, the culture medium was aspirated and the cells were washed with PBS. For extraction of total protein, cells were lysed in the buffer containing 0.2% Triton X-100 in 20 mM Tris-HCl on ice for 15 minutes. For 3D cultures, the Alg scaffolds were first exposed to 55 mM sodium citrate for depolymerization at 25 °C for 25 minutes. Then, the cells were lysed by adding the same lyses buffer. The cell lysate was centrifuged at 2500 g at 4 °C for 15 minutes, and the supernatant was used to assay ALP activity. Total protein was finally used for normalizing the enzyme activity level (U).
Alizarin Red S staining was applied to evaluate the calcium deposition. After 7, 14, and 21 days of differentiation, culture medium was aspirated, the 2D and 3D cultures were washed in PBS and fixed in 10% formaldehyde for 30 minutes. Following double washes with PBS, the samples were stained with 2 % Alizarin Red S (Sigma Aldrich) for 10 minutes. Additionally, quantitative analysis of calcium content was performed by adding 500 μL of 100 mM cetylpyridinium chloride monohydrate (Merck, Germany) to elute alizarin red S. Then absorbance was evaluated at 405 nm.
Confocal Raman microscope
After 21 days of differentiation, all 3D samples were lyophilized, and fractured into pieces, then, the Raman spectra of the cell laden scaffolds along with Shilajit were obtained by Lab-Ram HR Confocal Raman spectrometer (Horiba, Japan). Both Shilajit and 3D samples were excited with laser power level at 50 mW using the excitation laser with the wavelength of 785 nm with 100 mV power for Raman spectra recording. We analyzed the Raman shift at the range of 500-2000 cm−1.
Biodegradation test
The in vitro biodegradation test was carried out to evaluate the possible impact of Shilajit on the degradation rate of Alg hydrogel scaffolds. To do this, 1% Alg hydrogels were prepared in the present or absence of Shilajit and electrogellate by 2.5% CaCl2 (n =3). Prior to enzyme application, the scaffolds were weighed. Then 500 µL of 0.01% trypsin (Bioidea, Iran) was added, and incubated at 37 °C for 12, 24,48, 72,100 and 122 hours. At the selected time points, trypsin was completely removed and experimental plates weighed. The enzymatic degradation rate was calculated by subtracting the start and end weight of the scaffolds.
Scratch assay (wound healing assay)
ASCs were seeded at 3×104 cells/well and permitted to adhere and form a confluent. A scratch as a straight line through the center of each well was formed by a sterile 10 μl pipette tip and the wells were washed with PBS to remove cell debris. After that, 500μL of DMEM with or without Shilajit was added to each well as treated and untreated groups. The staining was done with crystal violet at zero, 8 and 12 hours after incubation. The cell migration to the scratch was evaluated in both treated and untreated groups using the Image j software(https://imagej.nih.gov/ij/index.html). The cell free area in each group was calculated at each point of time.
Statistical analysis
Graph Pad Prism software version 8.1 was used to analysis and depicted the graphs. One-way ANOVA and two-way ANOVA were applied for compare the data from multiple groups. The results were displayed as mean ± standard error mean (SEM)and p value < 0.05 was defined as significant. All experiments were performed in triplicate.