3.1. Preparation of Hydroxyapatite Nanoparticles
The nanostructured hydroxyapatite (nHA) powder was prepared by the sol-gel method (
7). Briefly, 0.5 mol of phosphorus pentoxide (P 2O 5, Sigma Aldrich) was dissolved per liter of absolute ethanol (Merck, Germany). Separately, 0.5 mol of calcium nitrate tetrahydrate (Ca(NO 3) 2.4H 2O, Sigma Aldrich) was dissolved per liter of absolute ethanol. A mixture of Ca/P (in a mole ratio of 1.67) was obtained by the homogenous mixing of the two prepared solutions. The solution was left at ambient temperature for 24 h to obtain a transparent solution. Next, the obtained gel was dried at 80°C. Then, the gel was heat treated (heating up to 600°C at a rate of 5°C/min for 0.5 h, followed by cooling down at ambient temperature). The obtained powder was ball-milled with zirconia vial and balls the capacity of vial and diameter of balls were 125 mL and 20 mm, respectively). 3.2. Preparation of Hydrogel
A solution was prepared to contain 2% (w/v) sodium alginate (biochemical grade, molecular weight = 398, Sigma Aldrich, USA) dissolved in distilled water under magnetic stirring at room temperature. Separately, 10% (w/v) polyvinyl alcohol (PVA, hydrolysis 99%, molecular weight = (30 - 50)×10
3, Sigma Aldrich) was dissolved in distilled water under magnetic stirring at 80°C for 12 h. The same volume of the two solutions was uniformly mixed together at ambient temperature for 24 h. The synthesized nHA was added to the PVA/alginate solution at the concentrations of 1% (w/v) and stirred at room temperature for 12 h to obtain a homogenous solution. Then, the solution was poured in 12-well plates. Physical cross-linking of PVA in the mixture was done by the modified freeze-thawing method ( 8). In this way, the well plates containing alginate/PVA/HA solution were exposed several times to the freeze-thawing cycle without using any cross-linking agent that are toxic to cells. To do this, the solution in wells was exposed several times to freezing and thawing at -40°C and 5°C, respectively. By this method, the original shape of the mixture gel disk was kept and any harmful crosslinking agent was removed. After that, the disk shape of alginate-PVA-HA hydrogel was immersed in 2% (w/v) CaCl 2 solution to chemically crosslink the alginate. The disks were washed three times with water and finally freeze-dried (Telstar, Spain). 3.3. Characterization of Prepared Scaffolds
The apparent and total porosity of the fabricated scaffolds were measured using the water displacement principle using the Archimedes method (ISO standard 39231/1-1979(E)) (
where W1, W2, and W3 are the weight of dry scaffold, wet scaffold when it was removed from the water after 24-h soaking, and soaked scaffold in the water, respectively. To evaluate the wettability property of the fabricated scaffolds, the sessile drop technique was used. In this method, the fabricated scaffolds were placed under the needle of a contact angle measurement machine. A distilled water droplet was placed on the surface of the scaffold. The contact angle of the formed water droplet on the surface was measured by Image J software (Image J 1.40 g). Each experiment was repeated three times. The compressive strength of the prepared scaffold was evaluated by Instron 5542 machine tester (Norwood, MA, USA). The surface and pore morphologies of the fabricated scaffold were observed by scanning electron microscopy (SEM, Philips XL30: Eindhoven, The Netherlands).
The average pore size was estimated by Image J software. Since blood plasma has different inorganic ions, such as Na
+, Ca 2+, and K +, and organic compounds, like amino acids, the evaluation of in vitro degradation of the prepared scaffolds in media containing different salts has a significant role in its biomedical application. Therefore, in this study, two different solutions were chosen in order to assess the degradation rate of the fabricated scaffolds, including Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) and the mixture of 90% DMEM and 10% Fetal Bovine Serum (FBS, Sigma Aldrich, USA). To do this, the scaffolds were sliced to the same dimensions (5mm × 5mm × 2mm) and immersed in the solutions. The immersed samples were maintained in a shaking incubator at 37°C during the whole experimental period. After 28 days, the samples were collected and gently dried at ambient temperature for 24h. The weight loss of the samples in each period was measured by the following equation:
where W1 and W2 are the weights of the scaffold before and after soaking in the solutions, respectively.
3.4. Periodontal Ligament Stem Cells (PDLSCs) Isolation and Culture
To evaluate the cellular behavior of the fabricated scaffolds, PDLSCs (extracted from the mature human teeth) were used. Briefly, after washing the intact teeth by phosphate buffered saline (PBS), periodontal ligaments were scraped from the root area. The obtained tissues were rinsed several times with PBS, crushed to small pieces, and put in an incubator at the temperature of 37°C and in the atmosphere containing 5% CO
After reaching 80% confluency, the cells were cultured in DMEM containing 10% (v/v) FBS, 100 units/mL of penicillin, and 100 mg/mL of streptomycin and were kept in a humidified incubator for further experiments.
3.5. Cell viability and Proliferation Assays
Cell culture experiments were carried out by seeding 1×10
4 PDLSCs on the surface of each prepared scaffold in a 96-well plate using DMEM + 10% (v/v) FBS + 100 unit/mL penicillin + 100 µg/mL streptomycin. These experiments were performed in a humidified incubator. The media were refreshed every 48 h. The cytotoxicity and viability of the cultured cells on the fabricated scaffolds were evaluated by 3-(4,5-Dimethylthiazol-2-yl)- 2,5-Diphenyltetrazolium Bromide (MTT) assay kits (Sigma-Aldrich, St. Louis, USA) according to the manufacturer’s instructions. The experiments were carried out three times for each group and the absorbance was read by an Awareness Technology microplate-reader. 3.6. PDLSCs Differentiation Into Osteoblast-Like Cells in Fabricated Scaffolds
Sterilized fabricated scaffolds were placed in 24-well plates and 2 × 10
4 cells were seeded in each well. The media containing DMEM/F12 + FBS 10%, 50 μg/mL ascorbic acid, 10 nM dexamethasone, and 10 mM β-glycerol phosphate were added to each well for 21 days to encourage the differentiation of PDLSCs. The media were replaced by fresh ones every three days. 3.7. Real-Time RT-PCR
The effects of the fabricated scaffolds on the expression of four main bone-related genes including osteopontin, collagen I, osteocalcin, and alkaline phosphate (ALP) were evaluated by the real-time reverse transcription (RT) polymerase chain reaction (RT-PCR). After 21 days of PDLSCs culturing on the surface of scaffolds, samples were washed using PBS. A Qiazol reagent kit was used to isolate the total RNA pursuant to the manufacturer’s instruction. Next, cDNA was fabricated by an M-MLV reverse transcriptase kit. The obtained cDNA was exposed to quantitative RT-PCR. The targeted gene expression was measured by SYBR-Green (TAKARA, USA). The used primer sequences are presented in
Table 1 for osteonectin, collagen I, osteocalcin, and ALP.
Table 1. The Order of Used Primers for Quantitative RT-PCR
Gene Forward (5'-3') Reverse (5'-3') COL (Ι) ATGGCTGCACGAGTCACACC CAACGTCGAAGCCGAATTCC OCN TGCTTGTGACGAGGTATCAG GTGACATCCATACTTGCAGG ALP CCTCGTTGACACCTGGAAG CTGGTAGTTGTTGTGAGCATAG OPN GCCGACCAAGGAAAACTCACT GGCACAGGTGATGCCTAGGA
Abbreviations: ALP, alkaline phosphate; COL (I), collagen I; OCN, osteocalcin; OPN, osteopontin.
3.8. Immunocytochemistry Analysis
After 21 days of osteoblast-like cells induction, the particular markers in differentiated cells were detected by immunocytochemistry (ICC) experiment. First, the cells were fixed by 4% paraformaldehyde (Sigma-Aldrich). Then, the cells were permeabilized using 0.2% Triton-X100 (Sigma-Aldrich) for 30 min, followed by blocking with 5% bovine serum albumin in PBS.
After 45 min, the cells were incubated overnight with primary antibody collagen I (mouse monoclonal AB; Abcam,1:200) and osteonectin (Rabbit Monoclonal AB; Abcam, 1:200). A secondary antibody (Alexa Fluor@488 donkey anti-mouse IgG and Alexa Fluor@594 donkey anti-rabbit IgG diluted at 1:500; Abcam) was used for one hour. After each step, the slides were cleaned with PBS and the staining of nuclei was carried out using 4',6-diamidino-2-phenylindole (DAPI, Sigma). Cells were examined by fluorescence microscopy (Olympus BX51, Japan).
3.9. Statistical Analysis
All experiments were repeated three times and the mean ± standard error of the mean was reported. Two-way ANOVA was employed to analyze the data. Significant differences were shown as *P < 0.05, **P < 0.01, and ***P < 0.001.