2.1. Materials
Taxifolin (C15H12O7, purity > 96%, Mw = 304.25 g mol-1) was gifted by Beckman-Kenko GMBH Company (Bassum, Germany). Halloysite nanotubes, methylcellulose (Methocel® MC low viscosity, 300-560 mPa.s at 2% in H2O (20 °C), 27.5-32% methoxyl basis), thiazolyl blue tetrazolium bromide (MTT), dexamethasone, β-glycerophosphate, ascorbic acid-2-phosphate, ethanol, glutaraldehyde, formaldehyde and hydrochloric acid were purchased from Sigma-Aldrich (USA). Trisodium citrate (TSC) and dimethyl sulfoxide (DMSO) were from Merck Chemical Co (Germany) and dialysis bags (Mol. Wt. cut-off ~ 12–14 kDa) were from Betagen Co (Mashhad, Iran). Persian gum (PG, an anionic plant polysaccharide) was bought in crystalline form with different colors from white to reddish brown from Reyhan Gum Persian Co (Iran). Then, to generate a homogenous powder, it was milled and sieved through mesh No. 60. Human osteoblast-like cell line (MG-63) was provided by the National Cell Bank (Pasteur Institute, Iran). Dulbecco's Modified Eagle Medium- Low Glucose (DMEM-LG), fetal bovine serum (FBS), streptomycin/penicillin and phosphate buffer saline (PBS) were provided by Bioidea (Iran). The alkaline phosphatase kit was from Pars Azmoon Company (Iran) and the highly pure RNA isolation kit was from Roche Company (Germany). AddScript cDNA Synthesis Kit was purchased from Addbio (Korea) and Syber Green PCR Master Mix (RealQ plus 2×) was purchased from Amplicon (Denmark). Primer genes (COL1A1, BGLAP and SPP1) were provided by Cinaclon (Iran). Ketamine and acepromazine were from the Alfasan Company (Netherlands). Moreover, deionized water (DIW) was utilized throughout the study.
2.2. Specifications of PG
In terms of solubility, PG consists of two parts: one is totally soluble (30%) while another one (70%) is insoluble, which forms a gel network (Dabestani et al. 2018). Its main elements are K+, Na+, Mg2+, Zn2+, Fe2+ and Ca2+ (Abbasi 2017). The datasheet analysis of PG by the providing company indicated the following composition for the used PG; carbohydrate: 91.3%, protein: 1.2%, fat: 0.2%, total ash: 2.16%. PG is constructed of galactose (1→3 linked β-D-Glap) and rhamnose units in the backbone and (1→6) linked β-D-Glap), (1→3) linked α-L-Araf residues in side chains (Dabestani et al. 2018; Molaei and Jahanbin 2018; Ghasemzadeh and Modiri 2020). According to GC/MS results, the main monosaccharides of PG are included arabinose and galactose in a 2:1 ratio and small quantities of other monosaccharides such as rhamnose, mannose and xylose (Fadavi et al. 2014). The existence of various functional groups in the structure of PG such as -CH2, -CH3, -COO-, C-O, C-H, hydroxyl, carbonyl, carboxyl and amide groups, seen in FTIR spectra (Dabestani et al. 2018), allows it to be easily crosslinked with other carbohydrate polymers like gelatin and gum tragacanth (Khodaei et al. 2020). According to the manufacturer, the used PG also has the following features: molecular weight of 4.6×106, surface tension of about 55 mN/m, specific rotation of -6.8, pH of 4.4, and viscosity of 48 mPa.s for 1% solution.
2.3. Preparation of TAX loaded HNTs (HNTs-TAX)
To encapsulate TAX inside HNTs, TAX (8 mg) was dissolved completely in ethanol (10 ml) using a magnetic stirrer. Then varying quantities of HNTs were mixed into the aforementioned solution to obtain the mass ratio of 1:10, 1:5, 1:2, 1:1, 2:1 and 5:1 of TAX: HNTs. This slurry was then sonicated for 10 minutes before being placed in a vacuum jar. The 30-minute cycles of vacuum and atmospheric pressure were applied on the suspension. This process was repeated for 3, 5, and 7 cycles to reach the maximum loading efficiency. Eventually, after 10 min centrifugation of the suspension at 10000 rpm, HNTs-TAX nanoparticles were separated, washed with deionized water and lyophilized for other studies. To calculate the encapsulation efficiency (EE) of TAX in nanoparticles, after loading procedure the amount of free drug in the supernatant medium was measured spectrophotometrically (UV-mini 1240, Shimadzu, Japan) at 290 nm. The EE% was calculated according to Eq. (1) (Pan et al. 2017):
Where M0 is the TAX content at the beginning and M1 is the unloaded TAX.
2.4. Characterization of HNTs –TAX
The morphology of untreated HNTs was determined through a transmission electron microscope (TEM, Philips, CM300) to examine the in-depth data.
The infrared spectra of HNTs, TAX and HNTs-TAX were recorded on a JASCO FT/IR-6300 Spectrometer using KBr pellet method (400-4000 cm-1) at the resolution of 4 cm-1.
The thermal properties of the prepared nanoparticles were obtained with a gas-tight thermal analysis system (NETZSCH, STA 449 F3 Jupiter, Germany) under a nitrogen atmosphere. For this purpose, 5 mg of samples were loaded in aluminum pans, hermetically sealed and then heated from 25 to 400 ◦C at a rate of 10 ◦C min-1.
2.5. Preparation of hydrogel samples
The MC hydrogel was prepared based on procedure previously described (Dalwadi and Patel 2018). Briefly, MC powder (1.5 w/v%) was added to heated deionized water under continuous stirring for 30 min. Then, MC solution was crosslinked by trisodium citrate solution with a final concentration of 5 w/v% accompanied by stirring in an ice water bath until a clear solution was obtained. Finally, the solution was kept at 4 °C before it was used. The MC/PG hydrogels were made in the same way, except that 0.25, 0.5, 1 or 1.5 w/v% PG was first dispersed in deionized water under stirring at room temperature to obtain a homogeneous solution. Optimum MC/PG gel was selected according to injectability, rheological and mechanical properties. Then, different amounts of HNTs (1, 3, 5, 7 w/v%) were added to the optimized MC/PG hydrogel. Before adding HNTs to a warm stirring MC/PG solution, they were sonicated in deionized water for 10 minutes. Based on rheological and mechanical properties, the best MC/PG/HNTs sample was chosen as a carrier for prepared HNTs-TAX nanoparticles.
2.6. TAX release study
The release profiles of TAX from HNTs-TAX and hydrogels were evaluated by transferring the nanoparticles dispersion or hydrogels into the dialysis bags. Then the bags were immersed in 20 ml of PBS (pH 7.4, 37°C) in falcon tubes and were placed in a shaking thermostatic incubator with a constant speed of 100 rpm. At each time interval, 500 µl of released medium was picked up and its absorbance was measured by a spectrophotometer at lmax = 326 nm. For each formulation, the sample devoid of TAX was used as the blank. The standard calibration curve of TAX in PBS was used to calculate the TAX released percent. The tests were made in triplicate for each sample and the cumulative amount of released TAX was measured (Kouhi et al. 2020).
2.7. Characterization of hydrogel samples
2.7.1. Mechanical tests
A standard testing machine (SANTAM, STM-20, Iran) operating in compression mode was used to determine the mechanical properties of the prepared hydrogels (Hejazi and Mirzadeh 2016). The hydrogel samples were frozen in a 24-well culture plate overnight and freeze-dried for 48 h in a freeze dryer (Martin Christ, Germany) at 0.001 mbar. The cylindrical samples (diameter = 15 mm and height = 10 mm) were then placed between compression platens and the crosshead rate was set at 0.5 mm/min. The compressive load was applied up to 50% of the initial height of the samples. For each scaffold, the test was repeated five times and the average of the measurements was reported as mean ± standard deviation.
2.7.2. Injectability test
The injectability of MC, MC/PG and MC/PG/HNTs samples was evaluated using a texture analyzer (Brookfield, CT3-4500, USA) according to a previous study (Kim et al. 2018). A syringe (10 ml with a diameter of 15.8 mm) was charged with the gel sample and placed between the machine crosshead and compression plate at room temperature. At a crosshead speed of 0.4 mm/s, the compression stress was applied to the syringe plunger and a maximum limit of 50 N was selected for the test since the larger forces are not practically suitable in a manual injection. For each sample, the test was repeated in triplicate and the maximum force in the plateau area was determined as a maximum injectability force.
2.7.3. Morphological analysis
The hydrogel samples were morphologically studied using SEM (Philips, XL30). The hydrogels were frozen in a 24-well culture plate overnight, and then freeze-dried (Martin Christ, Germany). Before imaging, the samples were gold-coated under a vacuum.
2.7.4. In vitro physiological stability
Pre-weighted dried hydrogels (Wd) were incubated in 20 ml of PBS (37°C, pH 7.4) until equilibrium swelling occurred to investigate the effect of PG and HTN content on swelling behavior. Samples were removed from PBS at various periods; wiped off to remove its excess water and the swollen hydrogels were weighted (Ws). The relative swelling ratio (%) was assessed according to Eq. (2) (Kumar et al. 2019):
To examine the degradation rate of hydrogels, pre-weighted samples in dried form (W0) were incubated in PBS (37°C, pH 7.4). After 1, 4, 7, 14 and 28 days of incubation, the specimens were freeze-dried and weighted (Wt) instantly. The weight loss percentage was calculated using Eq. (3) (Amiryaghoubi et al. 2020):
Each test was repeated three times and the results were reported as mean ± standard deviation.
2.7.5. Rheological properties of the hydrogels
All rheological measurements were conducted using a rheometer (Anton Paar, MCR 301, Germany) equipped with concentric cylinder geometry, CC27, using bob and cup with diameters of 26.66 and 28.92 mm, respectively. Primarily, oscillatory temperature sweep experiments were performed for MC solution and its mixtures with PG at constant 1 Hz frequency and 1% amplitude gamma in the temperature range between 25 and 40°C with a heating rate of 1°C/min. Subsequently, oscillatory amplitude and frequency sweeps were carried out at 25 °C to determine the structural characterization of hydrogels containing HNTs. The amplitude sweep tests were applied from the strain 0.1 to 100% with an angular frequency of 10 rad/s to define the linear viscoelastic (LVE) region. In accordance, frequency sweeps were performed at a strain value of 1% over an angular frequency range of 100 to 0.1 Hz.
2.8. Cell culture studies
The MG-63 osteoblast-like cells were cultured in Dulbecco's Modified Eagle Medium- Low Glucose (DMEM-LG) with 10 v/v% fetal bovine serum (FBS) and 1 v/v% streptomycin/penicillin in an incubator containing 5% CO2. Every three days the culture medium was replaced. To evaluate cell-sample interaction, before cell seeding the hydrogel samples were washed with phosphate buffer saline at 37°C and sterilized for 2 h under UV light. Then, hydrogels (100 µl) were poured into a 24-well culture plate in a disk shape and were allowed to form a physical gel at 37°C incubator. Consequently, cell suspension (300 µl, 1.5×104 cells) was carefully seeded on each hydrogel and tissue culture plate (TCP) as control and incubated for 1 h to enhance cellular penetration and adhesion. Then extra culture medium was added to each well up to the final volume of 1 ml and continued incubating for predetermined time.
2.8.1. Cell proliferation
Proliferation of MG-63 cells cultured onto the samples was examined using MTT mitochondrial reduction assay. At the specific times (1, 4 and 7 days of cell culturing), the medium was thrown away and the cell-seeded samples and control were incubated with 500 µl of DMEM-LG and 50 µl of MTT solution (5 mg/ml) for 4 h. After discarding the medium carefully, the dark blue formazan crystals were dissolved in 300 µl DMSO and were kept for 1 h at 37°C. Lastly, a certain volume of each well (100 µl) was transferred to a 96-well plate and its optical density (OD) was read at 490 nm using a microplate reader (Biotek Instruments, China). The relative cell survival (%) was calculated according to the Eq. (4) (Mokhtari et al. 2019):
Where Xsample, Xb and Xc were absorbance of the sample, DMSO (blank) and TCP (control), respectively.
2.8.2. Cell attachment
The morphology of MG-63 cells adhering onto the MC/PG/HNTs-TAX hydrogel was characterized utilizing SEM. After 7 days of cell culture in osteogenic medium (DMEM-LG medium containing; 10 v/v% FBS, 1 v/v% antibiotic solution, 100 nM dexamethasone, 10 mM β-glycerophosphate and 0.2 mM ascorbic acid-2-phosphate), the medium was removed and the samples were washed with PBS and fixed in 4.5% glutaraldehyde for 3 h. After rinsing with PBS, the cell-seeded hydrogel was dehydrated with a graded ethanol series, dried overnight and prepared for SEM imaging (Tian et al. 2008).
2.8.3. Alkaline phosphatase (ALP) assay
In order to measure the activity of ALP as a marker of bone mineralization, the conversion of para nitrophenyl phosphate to para nitrophenol was evaluated as previously reported (Farokhi et al. 2012). 1×104 MG-63 cells were seeded on each sterilized hydrogel placed in 12-well plates and cultured in osteogenic medium for up to 14 days. After 7 and 14 days, the medium was discarded and by following the ALP kit manufacturer's instructions (Pars Azmoon kit, Iran), ALP activity was measured using spectrophotometry at a wavelength of 405 nm.
2.8.4. Alizarin Red S (ARS) staining
The ability of MG-63 osteoblast-like cells to form mineralized nodules in the presence of hydrogels was determined by the ARS method based on a previous research study (Sedghi et al. 2020). Briefly, in a 12-well plate the cells were seeded on each samples at a density of 1×104 cells/well. After 14 days the osteogenic medium was aspirated, the cells were rinsed for three times with PBS (37°C, pH 7.4) and then were fixed in formaldehyde (3.6 v/v%) at 37 °C for 15 min. The fixed cells were stained by 2% Alizarin Red S with the pH of 4.1-4.3 for 15 min in an incubator. After washing the cell layer with deionized, calcium deposition was visualized by a microscope (Olympus, Tokyo, Japan) and images were recorded with a digital camera (Sony, DSC-H9, Japan).
2.8.5. Osteogenic gene expression using real-time polymerase chain reaction (RT-PCR)
Briefly, after 14 days of cell culturing, total RNA was extracted by a High Pure RNA Isolation Kit based on the manufacturer's protocol (Roche, Germany). The quality of total RNA extracted from each sample was determined by a nano-spectrophotometer (WPA, England). Complementary DNA (cDNA) of each sample was prepared using AddScript cDNA Synthesis Kit (Addbio, Korea) according to the manufacturer's instructions with oligo dT primers. Quantitative RT-PCR was carried out using Syber Green PCR Master Mix (RealQ plus 2×, Amplicon, Denmark) and the StepOne™ Real-time PCR Detection System (Applied Biosystems, Life technology, California, US). The primer sequences are shown in Table 1. The comparative Ct (ΔΔCt) was used to compute the relative gene expression.
Table 1 Sequences of the specific primers used for RT-PCR analyses of various gene expressions in osteoblasts
|
Reverse primer
|
Forward primer
|
Gene
|
5'-CAGATCACGTCATCGCACAAC-3'
|
5'-GAGGGCCAAGACGAAGACATC-3'
|
Collagen 1
|
5'-CCCTCCTGCTTGGACACAAAG-3'
|
5'-CACTCCTCGCCCTATTGGC-3'
|
Osteocalcin
|
5'-TGAGGTGATGTCCTCGTCTG-3'
|
5'-GCCGAGGTGATAGTGTGGTT-3'
|
Osteopontin
|
5'-GCACAGAGCCTCGCCTT-3'
|
5'-GTTGTCGACGACGAGCG-3'
|
Beta-actin
|
2.9. Animal study
The animal study was performed under the guidelines of the Ethics Committee of Isfahan University of Medical Sciences (Ethical code, IR.MUI.RESEARCH.REC.1398.590). Healthy male Wistar rats (~250 g) were obtained from Animal House, School of Pharmacy, Isfahan University of Medical Science, Iran for the femoral bone defect model in vivo. Animals were housed in cages under 12 h light/dark cycles with provided water and food. The animals were divided into four groups each containing 6 rats: 1) healthy group (without defect), 2) untreated group as control (with defect), 3) the group treated with MC/PG/HNTs hydrogel containing MG-63 cells (with defect), and 4) the group with MC/PG/HNTs-TAX hydrogel containing MG-63 cells (with defect). Under sterile conditions, after the rats were anesthetized by intraperitoneal injection of ketamine (75 mg/kg) and acepromazine (2.5 mg/kg), an 8-10 mm incision was made in the cranial femur, proximal part, and then the Tensor Fascia lata was cut with scissors, opening the muscles along their natural length to reach the left femur. Then, a hole with a 5 mm diameter was drilled under constant irrigation with saline solution to prevent overheating and it was filled with a hydrogel containing MG-63 cells at the density of 1×106 cell/sample based on experimental design. The rats were sacrificed 6 weeks after implantation and the defects were analyzed through radiography. All femurs were fixed in 10% formalin for 48 h and decalcified in 10% HCl for 12 h. The specimens were embedded in paraffin and sectioned at 7 µm thickness. At least four samples per group were stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT) and randomly chose for histological observation under a microscope (Olympus, Tokyo, Japan).
2.10. Statistical analysis
All data expressed as mean ± standard deviation were statistically analyzed using one-way ANOVA analysis followed by the Turkey's post-hoc test using GraphPad Prism Software (V.8) and p-value < 0.05 was considered as statistically significant.