2.1. HA powder preparation
After collecting the carp bones, they were boiled in water for about 90 minutes to remove the fats attached to them. Then, they were air-dried, completely. Subsequently they were crushed and placed in an electric furnace to sinter at 700 °C for 5 hours (20). To confirm the formed phases in the powder, x-ray diffraction (XRD) analysis was done (Bruker, D8-advance). Afterward, to obtain uniform nanoparticles, HA powder was ball-milled with Teflon cup and tungsten carbide balls for 2h using a planetary ball mill machine (Amin Asia, Iran). The rotation speed was 400 rpm with ball to powder ratio of 20:1. Scanning electron microscopy (SEM) images of the n-HA were provided to analyze the morphology and size of HA powders after ball mill using MIRA3, TESCAN. Figures 1a and 2 show the XRD pattern and SEM images of HA powder. The obtained XRD spectrum matched the hydroxyapatite reference card of 24-0033, and that reported in previous studies (29-31). The HA main peaks were appeared at 2θ around 31.8°, 32.2°, and 32.9°, which are associated with diffraction planes of (211), (112), and (300), respectively (Figure 1a). Furthermore, SEM images (Figure 2) showed that HA powder size became at nano-scale level after 2h of ball mill.
2.2. NaF powder
Sodium fluoride powder was purchased from SIGMA-ALDRICH, Germany with molecular weight of 41.99 g/mol and purity of >99%. XRD analysis (Bruker, D8-advance) of the powder is shown in Figure 1b. The obtained XRD pattern matched the NaF reference card of 36-1455 and that of previously reported data (32, 33). The results indicated were well crystallized structure of NaF with diffraction at 2θ of about 33.65°, 38.95°, 56.20°, 67.07° and 70.41°, respectively, from Miller indices of (110), (101), (201), (102) and (211).
2.3. n-HA scaffold preparation and characterization
For preparing disc-shaped scaffolds, a 5% poly vinyl alcohol (PVA) solution was prepared as a binder. Then, n-HA powder was mixed with PVA and poured into a special plastic mold and completely compressed. After removing the samples from the mold, they were sintered in an electric furnace at 300 ° C for one hour, then the temperature was raised to 1100 ° C, and remained for one hour. XRD spectrum (Bruker, D8-advance) and SEM images (Philips XL30, Netherland) were provided after scaffold sintering.
2.4. Loading of NaF on n-HA scaffolds
Disc-shaped scaffolds were divided into three groups for NaF loading based on the immersion time (5-minute, 7.5-minute, and 10-minute immersion). The NaF loading process was performed by placing the scaffolds in a 75 w/v% suspension of NaF in acetone on a stirrer. To prevent the evaporation of acetone, a lid was placed on the container having the suspension during processing. After the immersion time, the scaffolds were frozen at -20℃ and then lyophilized for 24h. The difference between the scaffold weights was calculated by weighing the scaffolds before and after NaF loading to estimate the amount of loaded NaF on the scaffolds.
SEM images (FE-SEM, Sigma 300-HV, Ziess, Germany) were provided after loading of NaF on the surfaces of scaffolds. First, the scaffolds were sputter-coated by a conductive material and then the images were taken at secondary electron mode. Moreover, elemental map analyses of the NaF loaded scaffolds was accomplished using energy dispersive X-ray spectroscopy (EDS).
2.5. NaF release profile
For evaluation of loading efficiency and NaF release, disc-shaped scaffolds that loaded with NaF have been put in dialysis bags filled with 2 mL phosphate-buffered saline (PBS). Each dialysis bags were placed in a container containing 10 mL of PBS, on a magnetic stirrer. In different time points (1, 3, 6, 24, and 48 h), 200 μL of the PBS was taken from the container to measure the optical density (absorbance) at 630 nm using a microplate reader (Synergy H1 Hybrid Multi-Mode Microplate Reader, BioTek, USA). After each sampling, an amount equal to the removed sample replaced with PBS. A standard curve of NaF concentration-optical density was obtained by measuring the absorbance of known NaF concentrations. This standard curve was used to convert the sample absorbance to concentration. The cumulative% of NaF release was then calculated and plotted as a function of time.
2.6. Scaffold sterilization and preparation of medium extract
For sterilization, the scaffolds were covered individually with aluminum foil and placed in glass sterile Petri dishes, which were kept in an oven at 170°C for 30 minutes. They were then transferred under a laminar cell culture hood for cellular tests. To prepare the extract medium, after sterilization, two samples from each group were immersed in complete culture medium (2.0 ml/well) in a 24-well plate for 3 days. Afterwards, the scaffolds were discarded and the culture media were used for the tests.
2.7. Cell culture
Dental pulp stem cells (DPSCs) were purchased from Royan Stem Cell Technology (Isfahan, Iran). Dulbecco's Modified Eagle Medium (DMEM, Gibco, Grand Island, USA) that contained 10% fetal bovine serum (FBS, Gibco, Grand Island, USA), Penicillin (100IU), Streptomycin (100 µg/mL) were used as a cell culture medium. The cells were grown with 95% humidity in the presence of 5% CO2 at 37 °C.
2.8. Cytotoxicity tests
Viability of the cells exposed to the scaffold extract medium (or scaffold cytotoxicity) was evaluated by a lactate dehydrogenase (LDH) cytotoxicity detection kit (Roche Applied Science, Germany). DPSCs cells (5×103 cells per well) were seeded in a 96-well plate in a complete growth medium (DMEM/F12+10% FBS) for 24h to form a monolayer. The medium was then replaced by scaffold extract media. After 3 and 7 days of incubation, the plate was centrifuged, and the supernatants were transferred to another 96-well ELISA plate. After that the LDH detection mixture was inserted into the plate wells (100 μL per well), which were then incubated at room temperature (30 minutes). The optical density was measured by a microplate ELISA reader at 490 nm (Synergy H1 Hybrid Multi-Mode Microplate Reader, BioTek, USA). The cell viability% was calculated by equations 1 and 2.
Where low control was a mixture of the supernatants of cells cultured without scaffold extract medium and the LDH detection mixture. Furthermore, high control was prepared by lysing the cells with triton X-100.
2.9. Alkaline phosphatase assay
DPSCs at a density of 2×103 cells per well in a 96-well plate were cultured in a complete culture medium for 24h to form a cell monolayer. The medium was then replaced by scaffold extract media, and osteogenic factors including ascorbic acid (vitamin C, 50 µg/mL), and β-glycerophosphate (10mM) (Sigma-Aldrich, USA) were also added. ALP activity was measured using SensoLyte pNPP Alkaline Phosphatase Assay Kit (AnaSpec, USA) according to the protocol provided by the manufacturer. Briefly, 7 and 14 days after cell culture with scaffold medium extracts, cells were homogenized in 100 μL lysis buffer provided with the kit. The obtained lysate was centrifuged for 15 min at 10000 g, 4º C. The supernatant was collected for ALP assay using p-nitrophenyl phosphate (p-NPP) as a phosphatase substrate (for 20 min at room temperature). The alkaline phosphatase standard was supplied with the kit. The absorbance was measured at 405 nm (Synergy H1 Hybrid Multi-Mode Microplate Reader, BioTek, USA).
2.10. Osteocalcin assay
DPSCs cells (5×103 cells per well) were seeded in a 96-well plate and cultured in a complete growth medium for 24h allowing cells to form a monolayer. The complete medium was then replaced by scaffold extract media. After 7 days of incubation, the osteocalcin level was assessed in the cell culture supernatant by a sandwich ELISA method using rat osteocalcin/bone gamma-carboxyglutamic acid-containing protein (OT/BGLAP) ELISA Kit (ZellBio, Germany) according to the instruction provided by the manufacturer.
2.11. Gene expression analysis by RT-PCR (qRT-PCR)
Assessment of bone-specific and angiogenic genes in the DPSCs cells cultured in scaffold extract media after 3 days including Runt-related transcription factor 2 (Runx2), type I collagen (COLI), and vascular endothelial growth factor (VEGF) involved in bone differentiation was performed using real-time polymerase chain reaction (RT-PCR).
Total RNA from cells was isolated using TRIzol reagent (Invitrogen). The cDNA was synthesized according to the protocol of the Revert Aid™ First Strand cDNA Synthesis Kit (Takara, Japan). cDNA was synthesized and the expression levels of Runx2, COLI, and VEGF mRNA were measured and quantified using the Corbett Life Science RT Detection System and the SYBR Green Master Mix Kit (Takara). B2M was the endogenous control used to normalize the mRNA expression levels. The primer sequences are shown in Table 1. This real-time quantitative PCR (RT-qPCR) approach allows precise measurement of gene expression, with B2M serving as a reliable internal reference for analysis. Our data were analyzed using the 2-ΔΔC method.
Table1: Primers sequence was used in RT- PCR
Sequence
|
Name
|
B2m
|
F: 5′-CGTGATCTTTCTGGTGCTTGTC-3′
R: 5′-GGAAGTTGGGCTTCCCATTCT-3′
|
Col1a1
|
F: 5′-AAGAGCGGAGAGTACTGGAT-3′
R: 5′-CTTGGGGTTTGGGCTGATGT-3′
|
RUNX2
|
F:5′-GGAACCAAGAAGGCACAGAC-3′
R: 5′-CGGGACACCTACTCTCATACT-3′
|
VEGF-A
|
F: 5′- TCGGAGAGCAACGTCACTA-3′
R: 5′- TCTTTGGTCTGCATTCACATC-3′
|
2.12. Cell adhesion test
For cell adhesion, DPSCs (2 × 103 cells per well) were seeded onto the scaffold surfaces directly and incubated at 37 °C, 5% CO2 and 95% humidity. After 3 days of incubation, the cells on the scaffolds were fixed with paraformaldehyde (4%) at 4℃ for 20 min, rinsed with PBS, dried, and gold-coated to observe under SEM (FE-SEM, Sigma 300-HV, Ziess, Germany) (FE-SEM, Sigma 300-HV, Ziess, Germany).
2.13. Statistical analysis
All data were analyzed using the analysis of variance (ANOVA) test using Minitab V17. P-values < 0.05 (confidence level 95%) were considered significant. Post-hoc analysis was performed via Tukey’s multiple comparison test.