1. Cells culture and osteogenic differentiation
Deciduous teeth were obtained from healthy children of 6–8 years old through a procedure authorized by the Ethics Committee of Liaocheng People’s Hospital (Liaocheng, China).The SHEDs were isolated from the dental pulp by outgrowth method as described in previous report[26]. For osteogenic induction, SHED cells were cultured in osteogenic induction medium for two weeks and the medium was replaced twice weekly. Osteogenic differentiation medium composition: 90% DMEM (Gibco), 10% fetal bovine serum (Gibco), 100 U/ml streptomycin (Gibco) and 100 U/ml penicillin (Gibco), 10 mM β‑glycerol phosphate, 100 nmol/l dexamesone, 50 µM l‑ascorbic acid 2‑phosphate and 50 nM vitamin D3 (Sigma‑Aldrich). Two weeks later after osteogenic induction, mineral deposit formation was identified by Alizarin Red S staining. The cells were first fixed with 4% paraformaldehyde (m/v; Sigma-Aldrich), and then stained with 40 mM Alizarin Red S (pH 4.2; Sigma-Aldrich) at room temperature for 15 min. Finally, images were captured with an inverted fluorescence microscope (Nikon Ti, Japan).
2. Western blot analysis
Total protein was extracted from osteo-induced cells with RIPA buffer and quantified using a BCA protein assay kit (Beyotime, Beijing, China). Total protein about 20μg extracted from osteogenic differentiation and control group were separated with 10% sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF)membrane (0.45μm, Milipore, Germy) using the Bio-Rad protein assay system (Bio-Rad, USA). Runx2 (1: 1000, Abcam, Cambridge, MA), Alp (1 : 1000, Abcam, Cambridge, MA), and 𝛽-actin (1 : 2000, Santa CruzBiotechnology, Santa Cruz, CA) were used as primary antibodies. Horseradish peroxidase- (HRP-) linked goat anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were used as the secondary antibodies. All bands were analyzed using Image Lab Software (version 1.6 NIH) to determine the relative level by comparing with the internal control β-actin.
3.Fluorescence microscope observation
Cells were trypsized and seeded in 6-well plates at the density of 2×104 cells/cm2. Molday ION Rhodamine B (MIRB, BioPAL Co., Worcester, MA, USA) stock solution (2mgFe/mL) was added to the normal culture medium (DMEM/F12 + 10% FBS) for preparing labeling medium with the final concentrations of 0, 12.5, 25, 50 and 100 𝜇gFe/mL respectively. SHED cells were incubated in labeling mediun for 24h under standard culture conditions (37℃, 5% humidified CO2). The cells were washed 3 times with PBS and then fixed with 4% paraformaldehyde for 15 min. After wash 3 times with PBS, the cells were blocked with 5% BSA dissolved in Triton X-100 for 1 hour. Then FITC-phalloidin conjugate solution (Enzo, New York, NY, USA) was used to stain Filamentous actin at room temperature for 1 hour. After washed 3 times with PBS, cells were observed with fluorescence microscope.
4. Prussion blue staining and labeling efficiency
After labeled with various concentration of MIRB, SHED cells were fixed with 4% paraformaldehyde. Wash 3 times with PBS. SHED cells were incubated with Perl's Prussian blue reagent (2% potassium ferrocyanide was dissolved in 6% hydrochloric acid) (Leagene Biotechnology, Beijing,China) for 30min. The distribution of intracellular Fe3+ was observed with light microscopy. In general 10 random fields of view were selected under a 10x microscope to count Prussian blue positive staining cells, and the labeling efficiency was calculated as following: Labeling efficiency = (number of Prussian blue positive cells/ number of total cells) × 100%.
5. Detection of intracellular iron content
The average intracellular iron content was measured with an iron assay kit (Biovision,Inc., CA, USA). The detailed detection method is as follows: First, SHED cells were seeded in a 6-well plate at the density of 2×104 cells/cm2 and incubate with MIRB labeling medium for 24h. Then labeled SHED cells were lysed with 65𝜇L Iron Assay Buffer. Centrifuge the lysed product at 16000g for 10 minutes. Add 50 𝜇L supernatant to the well of 96-well plate and add an appropriate volume of Iron Assay Buffer to make the final volume 100 μl. Add 5𝜇L iron reducer to each well for transforming Fe3+ into Fe2+. Add 100 𝜇L Iron Probe into each hole, and incubate for 1 h at room temperature. Absorbance was read with a spectrometer at 593nm. Meanwhile iron content standard curve was drawn according to the instructions in the kit. Calculate the total iron content in each hole according to the standard curve. Count the number of cells in each well with a hemocytometer. Iron content of a single cell = total iron content per well/number of cells per well.
6. Trypan blue staining and viable cells percentage calculation
Prepare 4% trypan blue stock reagent by adding 4g trypan blue (Merck KGaA, Darmstadt, Germany) to 100mL double-distilled water and filter with filter paper. Before use dilute to 0.4% with PBS. Five groups of cells in the control and labeled group were trypsinized to prepare single cell suspension and diluted appropriately. Mix 1 part of cell suspension and 1 part of 0.4% trypan blue and allow the mixture incubate at room temperature for about 3 minutes. Then count the live and dead cells separately (the microscope shows that the dead cells are stained light blue, and the viable cells are colorless after staining). Viable cells percent (%) = number of viable cells/(number of viable cells + number of dead cells) × 100%.
7. The effect of MIRB labeling on the proliferation of SHED cells
Seed SHED cells into the 96-well plates at the density of 1000 cells/well. The SHED cells are divided into 5 groups: 0, 12.5, 25, 50, 100μg Fe/mL respectively. There are 8 multiple holes in each group, each hole is 100μL. After SHED cells adhered to the wall, 4 groups of cells were labeled with different concentrations of MIRB. After 24h incubation, change to normal culture medium and continue culture for 1, 3, 5, and 7 days. Then add 10μL CCK-8 solution (Yeasen, Shanghai, China) to each well at each time point, incubate at 37°C for 4 hours, and detect the absorbance at 450nm with a microplate reader.
8. The effect of MIRB labeling on the osteogenic differentiation of SHED cells
SHED cells were trypsinized and seeded in a 6-well plate at the density of 2×104cells/cm2. When the cells reach 80% confluence, change the culture medium to MIRB labeling solutions with concentrations of 0, 12.5, 25 and 50μg Fe/mL respectively. After incubate for 24 hours, MIRB labeling medium was replaced with the osteogenic induction fluid, and the induction was terminated after 14 days. Fix the cells with 4% paraformaldehyde for 30 minutes and wash three times with PBS. Add 2mL Alizarin Red S solution (pH 4.2; Sigma‑Aldrich) to each well and stain for 15 minutes. Wash 3 times with PBS, take pictures and record the staining of each well. For semi-quantitative analysis of Alizarin Red S, add 600 𝜇L cetylpyridinium chloride solution (100g/L; Sigma‑Aldrich) to each well and incubate at room temperature for 15 min. Detect the absorbance of the supernatant at the wavelength of 562nm.
9. Magnetic Resonance Imaging of MIRB labeling SHED cells in vitro
SHED labeled with different concentrations of MIRB (0, 12.5, 25, 50μg/mL) were collected and counted with a hemacytometer. One million cells per group were transferred to 1.5ml centrifuge tubes (Eppendorf, Westbury, NY, USA) respectively. After centrifuged at 150×g for 5 min, SHED cells were resuspended in 15μL thrombin solution. Then the 15 μL thrombin solution containing 1×106 cells and 15 μL fibrinogen solution were injected into the bottom of the 1.5ml centrifuge tube at the same time. The mixture condensed into a jelly in 1-2 minutes. Then the tubes were imaged with a 1.5 T system (Achieva, Philips Healthcare, the Netherlands) with a 8-channel Wrist Coil(repetition time [𝑇 𝑅 ] =20ms, International time [𝑇 𝐸 ] = 8.1ms, flip angle = 25, field of view [FOV] =80 × 80×30mm 3 , slice thickness = 4mm, Mat: 208 × 208).
10. Animals and group design
Forty eight SD rats about 6-8 weeks old (200-250g, male) were used in our study. For investigating the effect of transplanted SHEDs on periodontal bone regeneration, all rats used in this research were numbered and divided into four groups randomly. Each rat has one defect at the right mandibles. The four group rats were: (1) SHED(MIRB labeled), (2) SHED, (3) Fibrin and (4) PBS. Six rats in each group were anesthetized at 0, 3, 6 and 9 weeks after the operation, and the defect healing was captured by MRI. At 2 and 4 weeks after operation, 3 rats in each group were perfused to obtain samples of periodontal bone defects for Histological and Immunohistochemical analysis.
11. Preparation periodontal bone defects in rats
Periodental bone defects were constructed as the following procedures. Rats were anesthetized with 10% chloral hydrate (at the dose of 4ml/kg) by intraperitoneal injection. An extra-oral incision about 2cm long was made which was parallel to the inferior edge of rat mandibles. Separate the subcutaneous tissues and masseter muscles from the surface of mandible. A periodontal wound defect (length×height×depth: 5mm×4mm×1mm) was constructed with a dental drill at a low speed accompanying with lasting irrigation of physiological saline. The defect was located at 1mm behind the front of the mandible and 1mm below the crest of the alveolar bone. Muscle and skin were sutured separately and the wound was clean with Iodophor after the surgery. In order to avoid being rejected of the transplanted cells by the immune system, the rats were injected with cyclosporine A (10 mg/kg; Sigma) intraperitoneally one day before transplantation and every day after transplantation until the rats were sacrificed.
12. Preparation of fibrin glue containing SHED cells for transplantation
Fibrin glue was prepared using a Fibrin sealant kit (SHANGHAI RASS, Shanghai, China) according to the instructions of manufacturer, by ejecting equal volumes (15 μl) of a fibrinogen solution (40 mg/ml) and thrombin solutions (450 IU/ml) through a two-channel syringe. For cell transplantation, one million SHED fresh cells (MIRB labeled of unlabeled) were resuspended with 15μL thrombin solution. After preparation of periodontal bone defects in rats, the thrombin solution containing SHED cells (labeled or unlabeled) were injected into the defect region along with equal volume fibrinogen solution at the same time. In Fibrin group only fibrin glue without SHED cells and in PBS group only equal volume PBS was injected to the defect region.
13. MRI of MIRB-Labeled SHED in vivo
MRI of anesthetized rats was performed at 0, 3, 6 and 9 weeks after transplantation. All rats undergoing MRI testing were performed on a 1.5 T system (Achieva, Philips Healthcare, the Netherlands) using an 8-channel wristband coil. Rats were positioned head forward in a prone position. The axial and coronal three-dimensional fast field echoT1-weighted imaging“black bone” sequence was scanned. Sequence specifications are listed in Table 1.
Table 1. The dedicated parameters for the 3D T1 FFE sequence.
Axial 3D T1 FFE
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Acquisition time 4:44min ( 02:48 min and 01:56min respectively)
FOV 80 ×80×30mm3
Matrix 208× 208
Voxel size 0.385 × 0.385 × 0.75mm3
Acq voxel 0.385 × 0.385 × 1.50mm3
REC voxel 0.16 × 0.16 × 0.75mm3
Reconstuction Matrix 512
Number of signal averages 2
TR 20 ms
TE 8.1ms
Flip angle(deg) 25
WFS (pix)/bandwidth (Hz) 1.004/216.3
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14. MRI Image Analysis and Quantification
For image analyzing, the obtained DICOM datasets were reconstructed in the axial and coronal plane. The reconstruction slice number was 40 and the thickness was 0.5 mm. The qualitative and quantitative evaluations were performed separately by two radiologists certified by the Radiology Committee without interfering with each other. Imaging parameters comprised the defect area in defined regions of interest (ROIs) and signal intensity value.
15. Samples collection and preparation for histological and immunohistochemical analysis
Rats were sacrificed at 2 and 4 weeks after operations. First rats were anesthetized with chloral hydrate (at the dose of 4ml/kg) and then perfused with 200ml normal saline and finally fixed with 200ml 4% paraformaldehyde. The right mandible of each rat was excised and fixed with 4% PFA for 24h. Then these mandibles were decalcified in 10% ethylenediaminetetraacetate (EDTA) at room temperature for 4 weeks. The decalcification solution was replaced twice a week. After decalcification is completed, the bone tissue was dehydrated with gradient ethanol and immersed in paraffin wax for 2hours. The tissue was embed in paraffin and cut into sections of 3-4μm thick. These paraffin sections were placed in a 62℃ oven for 20min for HE, or 2hours for immunohistochemical staining.
16. Histological analysis
In order to evaluate the regeneration of periodontal tissue in each group, every fifth slices were selected and stained with hematoxylin and eosin kits(Beyotime, China). Then the specimens were observed under Nikon Ti microscope (Nikon,Japan) and the area of newly formed bone was measured by Image Pro-plus 6.0 software (Silver Spring, USA). The regeneration of defect area was calculated with the flowing equation: Percentage of bone regeneration = area of newly formed bone /area of total defect×100%.
17. Immunohistochemical staining
The osteogenesis of the mandibular defect was estimated by immunohistochemical analysis of Runx2 and Ocn. The paraffin sections were dewaxed with xylene treatment and rehydrated in gradient ethanol. Immunohistochemical staining was performed with Biotin-Streptavidin HRP Detection Systems (ZSGB-BIO, Beijing, China) according to the instruction of the kit. Briefly, to inhibit endogenous peroxidase, the sections were incubated with 3% hydrogen peroxide for 10 min. To block nonspecific staining, the sections were blocked in 1% goat serum for at least 30 min. Then these sections were incubated with primary antibodies of Runx2 (1:1000) and Ocn (1:500) (Abcam, Cambridge, MA, USA) for 2 hours at room temperature. Negative controls were incubated with PBS instead of the primary antibodies. The sections were then incubated with biotinylated goat anti-mouse or goat anti-rabbit immunoglobulin G (IgG) as a secondary antibody at 37°C for 15 min. The Immunoreactions were detected with diaminobenzidine (DAB) substrate kit (Maxim biotechnology Co., Ltd., Fuzhou, China). After being rinsed with running water for 5 min, the sections were counterstained with hematoxylin for 30 sec. Images were taken with a Nikon microscope (Nikon, Japan). Integrated optical densities (IOD) of Ocn and numbers of Runx2 positive cells in four groups were measured or counted using the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
18. Prussian blue staining of iron ions in vivo
Prussian blue staining was performed according to the instruction of the staining kit (Leagene Biotechnology, Beijing,China). Briefly, mix the A1 solution (2% potassium ferrocyanide) and A2 solution (6% hydrochloric acid) in the kit in a 1:1 volume to form the Perl’s staining solution. After the paraffin sections are deparaffinized and rehydrated, add Perl’s staining solution and incubate at room temperature for 30 minutes. Rinse three times with PBS, and finally counter-stain with solution B (nuclear fast red) in the kit for 5 minutes. Rinse three times with PBS, mount the slide after dehydration, and observe the staining under a microscope.
19. Statistical analysis
Data were expressed as the mean ± standard error of the mean and all the data were analyzed using SPSS software (SPSS Inc., IL, USA). Statistical significance was tested by Independent-Samples 𝑡-test. P < 0.05 was considered statistically significant.