Extraction of HA and characterization
First, the diaphysis of the cow's femur was boiled for 2–3 hours followed by washing in water to remove the excess tissue, cut into small pieces, delipidized in acetone, and dried overnight at 100°C. Extraction of HA was done by thermal decomposition method and using electric furnace (LAC, Ltd, Czech Republic) at 850°C for 3 hours; the powder obtained was pulverized by Planetary Ball Mill (Mpm-191, Iran). The X-ray differentiation method (XRD, Bruker, USA) was used to evaluate the purity of the extracted powder by comparing it with commercially purchased HA powder (Merck). CuKα beam was used for analysis. The scanning rate was equal to 1.54 degrees per minute and the diffraction angle was from 10 to 90 degrees. Also, to measure the particle size, we used SEM analysis of the extracted powder alongside Image J software (http://imagej.nih.gov/ij/index.html).
Scaffold construction
First, 6g of HA powder was added to 14 mL of distilled water and mixed on a stirrer for 5 min. An equivalent of 7% of HA powder (0.42 g) of triethyl phosphate (TEP; (C2H5)3PO4) (Sigma-Aldrich, USA) was added to the mixture and slurry was mixed on the stirrer for 24 hours. Then, an equivalent of 7% of HA powder (0.42 g), of glycerin (Sigma-Aldrich, USA) was added and stirred for 1.5 hours. Polyvinyl alcohol (PVA) (Sigma-Aldrich, USA) was prepared at a concentration of 15% and an equivalent of 33% of the weight of HA powder (2 g) was added to the slurry and mixed for 24 hours. Polyurethane foam was used as a template and cut to the desired size and shape. They were immersed in slurry and dried at 90°C for 15 minutes. The immersing and drying periods of the foams were repeated twice, and finally the slurry-coated foams were heated at a rate of 2°C/min to reach the temperature of 800°C; then, they remained at a temperature of 800°C for 5 hours. After that, the temperature increased at a speed of 3.5°C/min to reach the temperature of 1250°C and led them to sinterized for 3 hours. The temperature of the device was then dropped to ambient temperature.
The scaffolds were immersed in a 1% alginate solution (Sigma-Aldrich, USA) dissolved in phosphate-buffered saline (PBS) with/without TQ (Sigma-Aldrich, USA). To avoid trapping air bubbles, we placed them inside the vacuum pump machine for 10 minutes. Then, the gelation was done by adding 1% calcium chloride solution (Merck, Germany).
Characterization of the scaffold
SEM images were prepared by lyophilizing TQ free scaffolds. Gold replica was prepared using Q150R- ES sputter coater (Quorum Technologies, London, UK) and imaged using an VEGA3 microscope (TESCAN, Brno, Czech Republic) at 10 kV accelerating voltage. The pore size was estimated by analyzing SEM images using image J software. The porosity was measured by liquid exchange method.
The degradation rate was measured by incubating the scaffolds in 0.01% trypsin (Bioidea, Iran) and weighting them on days 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, and 56.
The hydration rate was evaluated by immersing lyophilized TQ loaded scaffolds in distilled water and weighting them at the beginning as well as at intervals of 5, 15, 30, 60, 120, and 240 seconds.
For evaluating the impact of TQ loading on mechanical strength, the scaffolds at a size of 10×10×3 mm3 and the same porosity were exposed to a mechanical resistance determination device (Zwick/Roell, Germany) at a speed of 0.5 mm/min.
TQ releasing assessment
To evaluate the drug release, we prepared the scaffolds according to what was mentioned in the previous part. Then, 1 mL of distilled water was added to each scaffold and the supernatant was removed at 20, 40, 60 seconds, 20, 40, 60 minutes, and 2, 4, 6, 12, 24, 48 hours. Finally, the release of TQ was assessed using the High-Performance Liquid Chromatography (HPLC AZURA; KNAUER, Germany) with a C18 column. The mobile phase consisted of a solvent system of acetonitrile and water with a ratio of 45:55. The flow rate was performed at 1 mL/min at 30°C and detection wavelength was at 254 nm.
Isolation, culture, and characterization of hADMSC
Adipose tissue samples from female patients undergoing liposuction under the age of 27–29 years were transferred to the laboratory under sterile conditions. The adipose tissue was cut into small pieces and treated with 0.2% collagenase type I (Gibco) in the serum-free medium for 45 minutes; then, the suspension was centrifuged for 5 minutes at 1500 rpm and the cell pellets were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) containing L-glutamine (1mM), Pen-Strep (1%) and 20% fetal bovine serum (FBS) (Gibco) and incubated at 37°C and 5% CO2. After 48 hours, the float cells were discarded, and the culture media was changed.
To characterize the hADMSCs, the cells were exposed to Fluorescein-5-isothiocyanate (FITC)-conjugated CD144 and CD44, to Phycoerythrin (PE)-conjugated CD34 and CD106, and to Peridinin-Chlorophyll-protein (PerCP)-conjugated CD105 antibodies all from Abcam. Finally, the percentage of expression of these CD markers was determined by flow cytometry using Flow Cytometer laser 488 nm (Becton Dickinson, NJ, USA), and Flowjo software was employed for analysis.
Differentiation of ADMSCs
The cells were aliquoted into 6 groups. The aliqoutes at a density of 1×106 cells per mL were mixed with alginate with or without TQ and OM and seeded into the HA scaffolds and gelation induced by adding CaCl2. Two of them were cultured on a 2D thin alginate coat with or without OM for 7 and 28 days. OM included 0.1 mM dexamethasone, 50 µM ascorbic acid, and 10 mM beta-glycerophosphate (Sigma-Aldrich) in DMEM medium. The culture media with or without OM contained 10% FBS (Gibco). The medium was changed every three days.
Cytotoxicity assessment
The 3-(4,5 dimethyl-2-thiazolyl) -2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma-Aldrich, USA) was performed at 3, 5, and 7 days after cell seeding to determine the appropriate and non-toxic dose of TQ and cell viability on the scaffolds. Briefly, to do this, TQ was prepared in 1% alginate at concentrations of 25, 50, and 100 µM, Then, 3×104 MSC/well was added to the alginate; they were impregnated into the HA scaffolds and allowed to be jellified by adding CaCl2. After 3, 5, and 7 days, the culture media were replaced with 500 µL of 0.5 mg/mL MTT and incubated at 37°C for 3 hours. The MTT was then eluted from the vital cells by adding 300 µL of dimethyl sulfoxide (DMSO; Merck) and incubating for 30 min. The optical density (OD) of eluted MTT was measured using a POLARstar Omega Plate Reader Spectrophotometer at 595 nm.
Cell differentiation assessments
To be sure about the presence of viable cells after a 28-day period of differentiation, we performed another MTT assay as described previously. The cell phenotype on the scaffolds after differentiation was obtained by SEM image.
Alkaline Phosphatase Activity
The alkaline phosphatase (ALP) activity was evaluated on days 7, 14, 21, and 28 of differentiation using a commercial kit (Pars Azmun, Iran) as a functional test for the osteoblasts. The basis of this kit is the conversion of colorless nitrophenyl phosphate to yellow nitrophenol and it was done according to the manufacturer's instructions. The yellow intensity of nitrophenol was measured with a plate reader spectrophotometer at 405 nm.
Alizarin Red S staining
Alizarin red S staining was used to evaluate the amount of calcium deposition by the osteoblast cells. After 28 days of differentiation, 2D and 3D cultures were fixed with 4% PFA for 20 min. The samples were exposed to a 1% Alizarin red S solution (Sigma-Aldrich) for 30 minutes. The sedimented dye was then eluted with 500 µl of 100 mM Cetyl pyridinium chloride monohydrate (Merck) and its optical density was measured with a plate reader spectrophotometer at 405 nm.
Cell attachment test
First, 3×104 cells were mixed with 1% alginate with/without TQ and seeded on the scaffolds. Then, 1% calcium chloride solution was added on them to start gelation of alginate and they were incubated in a serum-free environment for 2 hours. The scaffolds were then placed vertically in a falcon tube and centrifuged at 30 rpm for 30 minutes. The number of detached cells, as pellet, were then counted using a Neubauer chamber and subtracted by the number of initial cell number.
Real-Time qRT-PCR
Total RNA from differentiated osteoblast was isolated using the TRIzol (YTzol pure RNA, CinnaGen, Tehran, Iran), according to the company’s instructions. Extracted RNA content was quantified by NanoPhotometer (NanoDrop 2000, Implen, Germany). cDNA was produced with the aid of a High-Capacity RNA to cDNA kit (Thermo Fisher Scientific, USA), according to the manufacturer’s protocol. The expression of Collagen Ι, Osteocalcin and Osteopontin was evaluated by qRT-PCR (ABI7900HT, USA Specific primer sequences, as depicted in Table 1. PCR conditions were as follows: 10 min of initial denaturation at 94°C, followed by 40 cycles of denaturation at 95°C for 15 s; annealing was conducted at 60°C for 45 s, followed by 30 s extension at 72°C, and 5 min final extension at 72°C. PCR results were quantitatively analyzed by Rotor-Gene Q Series Software. For data analysis, the fold change (FC) of each gene expression was calculated by using the 2−ΔΔCt method, whereas housekeeping genes, β-actin and GAPDH, were used as controls to normalize the results.
Confocal Raman microscope
After 28 days of differentiation, all the 3D samples were fixed by 4% PFA for 20 min and then they were lyophilized; the samples were evaluated by Lab-Ram HR Confocal Raman spectrometer (Horiba, Japan). The spectra were prepared with an excitation wavelength of 633 nm and a maximum power of 17 mW from different parts of the scaffold, including filled pores with alginate, HA blades, and cells. To include organic and mineral excitations, Raman shift was investigated at the range of 500–2000 cm− 1.
Optical microscope
After 28 days of differentiation, the 3D groups were fixed using 20% PFA for 20 minutes and prepared for paraffin-embedded light microscopy. Due to the presence of HA in the samples and in the absence of demineralization during tissue preparation, the blocks were placed in 8% HCl and 8% formic acid from the starting of tissue cut point, to prevent the scaffolding from pulverizing during cutting. The samples were sectioned at 20 µm thickness, and stained with hematoxylin and eosin (H&E) and examined by a light microscope (Nikon E-200 microscope, Japan) and imaged.
Statistical analysis
Data are presented as mean ± standard deviation (SD). GraphPad Prism software (version 8.1) was used to quantitatively analyze the data and draw the graphs, and the data were analyzed using one way-ANOVA and two way-ANOVA statistical tests. P-value with less than 0.05 was considered as significant data. All experiments were performed in triplicate.