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Calcitonin gene-related peptide regulates periodontal tissue regeneration – Scientific Reports

Reagents

We used recombinant mouse CGRP (Phoenix Pharmaceuticals, Burlingame, CA, USA) for the experiments.

Cell culture

We established a mouse periodontal ligament cloned cell line, i.e., MPDL22 cells, and maintained it as described previously50. MPDL22 cells was grown in α-modification of Eagle’s medium (Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Biowest, Nuaillé, Pays De La Loire, France) and 60 g/mL kanamycin (Wako Pure Chemical Corporation, Osaka, Japan). Eagle’s medium (Wako Pure Chemical Corporation) (hereinafter mentioned as “culture medium”) with 100 ng/mL of human recombinant FGF-2 (Kaken Pharmaceutical, Tokyo, Japan) was added, and the cells were cultured under 5% CO2 at 37 °C and 95% humidity. For passaging, the cells were treated with phosphate-buffered saline (PBS) (Wako Pure Chemical Corporation) supplemented with 0.05% trypsin and 0.02% ethylene diamine tetraacetic acid (Thermo Fisher Scientific, Waltham, MA, USA) and seeded into 100 mm-culture dishes (Corning, Corning, NY Cells from the 37th to 40th generations of MPDL22 cells were used in the experiments.

Induction of differentiation

To induce differentiation of MPDL22 cells into hard tissue-forming cells, the cells were seeded into 12-well cell culture plates (Corning) at 1.0 × 105 cells/well and incubated in culture medium supplemented with 10 mM β-glycerophosphate (Wako Pure Chemical Corporation) and 50 μg/mL ascorbic acid (Wako Pure Chemical Corporation) (herein referred to as calcification). The culture medium was changed every 3 days thereafter.

Total RNA extraction

Total RNA extraction was performed using the nucleic acid extraction reagent RNA-Bee™ (TEL-TEST, Friendswood, TX, USA). RNA-Bee was added to the cells collected at the end of the culture to solubilize them. After adding 1/10 volume of chloroform (Wako Pure Chemical Corporation), the cells were centrifuged at 12,000 rpm for 15 min. The separated aqueous layer was collected, and RNA was precipitated by the addition of isopropanol (Wako Pure Chemical Corporation) and washed with 75% ethanol (Wako Pure Chemical Corporation). The total RNA precipitates were then dissolved in 20-μL diethylpyrocarboxylic acid-treated water (Wako Pure Chemical Corporation, hereafter abbreviated as DEPC-treated water), and the total RNA amount was measured using a NanoDrop ND-1000 (Thermo Fisher Scientific).

Complementary DNA (cDNA) preparation

cDNA was prepared by reverse transcription using total RNA as the template, and 1 μg of each RNA sample heat-treated at 65 °C was mixed with 20% five First Strand Buffer (Thermo Fisher Scientific), 1 mM dithiothreitol (Life Technologies, Gaithersburg, MD, USA), and 1 μg of each RNA sample heat-treated at 65 °C. The final concentration shown below was added to the sample: (Gaithersburg, MD, USA), 1.1-U/µL ribonuclease inhibitor (Takara Bio, Shiga, Japan), 0.5-mM dNTP mixture (Takara Bio), 5-U/µL Moloney-Mouse leukemia virus reverse transcriptase (Life technologies), and 55 ng/µL random Hexamers (Pharmacia Biotech, Milwaukee, WI, USA). The reaction solution was stored at 37 °C for 60 min and subsequently treated at 99 °C for 5 min to inactivate the residual enzymes for cDNA preparation.

cDNA amplification and detection by polymerase chain reaction (PCR)

The CGRP receptor is a dimer consisting of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). RAMP1 is a dimer composed of CLR and RAMP1, which, together with receptor component protein (RCP), drive the intracellular signaling pathway51.

For the expression analysis of Ramp1, Clr, and Rcp in MPDL22 cells, synthesized cDNA was used as a template and amplified by PCR using primers specific for each gene. AmpliTaq Gold™ 360 Master Mix (Thermo Fisher Scientific) 12.5 μL and 1 μL of cDNA solution, and 0.2 μM of each gene-specific primer (Gene Design, Osaka, Japan and FASMAC, Kanagawa, Japan) listed in Table 1 were added at a final concentration. The reaction solution was prepared with DEPC-treated water to a total volume of 25 μL. After heat treatment of the prepared reaction solution at 95 °C for 2 min, cDNA was amplified by Mastercycler®nexus GX2 (Eppendorf, Eppendorf, Hamburg, Germany) for 40 cycles of thermal denaturation at 95 °C for 30 s, annealing at 56 °C for 3 s, and extension reaction at 72 °C for 1 min as a single cycle. Forty cycles of cDNA amplification were performed. These PCR products were electrophoresed on 1.5% agarose gel (Wako Pure Chemical Corporation) and visualized using ethidium bromide (Wako Pure Chemical Corporation).

Table 1 Primers for RT-PCR.

Western blotting

Whole cell fractions obtained from MPDL22 cells were used for analysis of RAMP1 and CLR protein expression, and β-actin was used as a control. The total cell fraction was reduced by heat treatment with Laemmli’s 5 × sample buffer containing 2-mercaptoethanol (Wako Pure Chemical Corporation) at 98 °C for 10 min and then incubated with Mini-PROTEAN® TGX Precast Gel (Bio-Rad Laboratory) and Mini-PROTEAN® Tetra System (Bio-Rad Laboratory). PROTEAN® Tetra System (Bio-Rad Laboratory), and the protein lysate fraction was developed. The proteins were subsequently transferred to a membrane using Trans-Brot® Turbo (Bio-Rad Laboratory) and PowerPac™ (Bio-Rad Laboratory) (room temperature, 100 V, 3 h). Membranes were blocked (room temperature, 5 min) using Bullet Blocking One for western blotting (Nacalai tesque, Kyoto, Japan) and then probed with the following primary antibodies: rabbit monoclonal anti-RAMP1 (1:1000; Abcam, Tokyo, Japan, #ab156575) and mouse monoclonal anti-β-actin (1:5000; Sigma-Aldrich, #A5316). After washing, membranes were incubated with secondary antibodies, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG antibody (1: 5000; GE Healthcare, Chicago, IL, USA) or HRP-conjugated sheep anti-mouse IgG antibody (1: 10,000; GE Healthcare) and made to react with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) to amplify the luminescence signal; bands were detected with an ImageQuant LAS 4000 (GE Healthcare). We used Precision Plus Protein Dual Color Standards (BIO-RAD, #161-0374) as a ladder.

Real-time PCR

Real-time PCR analysis was performed using the gene-specific real-time PCR primers (Gene Design and FASMAC) listed in Table 2 with cDNA as the template (Biosystems, Foster City, CA, USA) on a Step One Plus Real-time PCR System® (Applied Biosystems). The expression levels of each gene were calculated relative to the expression level of the endogenous control gene, Hypoxanthin guanine phosphoribosyl transferase (Hprt), which is one of the housekeeping genes.

Table 2 Primers for real-time PCR.

Examination of calcified nodule formation ability by alizarin staining

The ability to form calcified nodules was examined by alizarin red staining. After induction of differentiation of MPDL22 cells into hard tissue-forming cells for 18 days, the culture supernatant was removed, the cell layer was washed twice with PBS, and fixed with 100% ethyl alcohol (Wako Pure Chemical Corporation) at 4 °C for 10 min. (Wako Pure Chemical Corporation) aqueous solution (pH 6.4) (room temperature, 5 min), followed by washing with distilled water. The stained images were scanned using a color image scanner GT-X970 (Epson, Tokyo, Japan). The images were then analyzed using WinRoof image analysis software (Mitani Corporation, Fukui, Japan). The fraction of stained area and color density were quantified, and the product was calculated as the degree of calcified nodule formation.

Experimental animals

Fifteen 8-week-old male BALB/c mice and 15 Ramp1 gene-deficient (Ramp1−/−) mice34,35 (weighing 17–21 g) were used for the experiments. These mice were generated, and heterozygous knockout mice were backcrossed to BALB/c mice for 12 generations. The 15 mice were divided into five groups of three mice each at 0, 3, 7, 10, and 14 days after silk ligation. They were maintained under controlled temperature (23 ± 2 °C) and light–dark cycle with free access to food and water and fed a regular chow diet (5.1% fat, 55.3% carbohydrate, 23.1% protein; MF Oriental Yeast Co., Ltd., Tokyo, Japan).

All the experiments in this study were performed in accordance with the animal experiment guide established by the Animal Experimentation Committee of the Graduate School of Dentistry, Osaka University (Animal Experimentation Committee of the Graduate School of Dentistry, Osaka University, Reception No.: Animal Dentistry R-01-019-0, Genetic Recombination Approval No.: 04484). All animal experiments complied with the ARRIVE guidelines. All efforts were made to minimize suffering.

BALB/c mice were purchased from Japan SLC Corporation (Shizuoka, Japan); Ramp1−/− (BALB/c genetic background) mice were obtained from RIKEN BRC (#RBRC0285)34,35. On the advice of RIKEN BRC, BALB/c mice were used as experimental controls.

Ligature-induced periodontitis model

We used a triple anaesthetic mixture of 75 µg/mL medetomidine hydrochloride (Nippon Zenyaku Kogyo, Tokyo, Japan), 400 µg/mL midazolam (Sandoz, Tokyo, Japan) and 500 µg/mL butorphanol tartrate (Meiji Seika Pharma, Tokyo, Japan). For awakening from anesthesia, an anesthetic antagonist (75 μg/mL atipamezole hydrochloride) adjusted with antisedan (Nippon Zenyaku Kogyo) and saline solution (Otsuka Pharmaceutical) was used.

Ligation and removal of silk threads were performed under deep anesthesia by intraperitoneal administration of 0.2 mL of the three anesthetic mixtures. After each procedure, the mice were awakened by intraperitoneal administration of 0.2 mL of the anesthetic antagonist. The mouse ligature-induced periodontitis model was performed according to Abe et al.52. The silk threads used were 5-0 PERMA-HAND SILK BLACK BRAIDED (Ethicon, Raritan, NJ, USA) and ligated to the maxillary bilateral second molars upon visualization under a binocular stereomicroscope.

Micro computed tomography (μCT)

Micro tomography images of the specimens were obtained using R-mCT2 (Rigaku, Tokyo, Japan) at a tube voltage of 90 kV, tube current of 160 μA, and slice width of 5 μm. The data obtained from the imaging were processed using three-dimensional image analysis software TRI/3D-BON (RATOC System Engineering, Tokyo, Japan), and two-dimensional images were obtained by setting the Z axis so that the X and Y coordinates of the contours of the maxillary left and right molars overlapped.

The two-dimensional images were imported into the image analysis software WinRoof (Mitani Corporation). The distance from the cemento-enamel junction at the center of the root to the alveolar crest was measured for the four roots: the distal root of the maxillary first molar, the mesial and distal roots of the maxillary second molar, and the mesial root of the maxillary third molar, and the total was evaluated as Total Bone Loss (Fig. 4b).

Preparation of the tissue sections

A catheter was inserted into the heart of 8-week-old mice that had been euthanized with CO2 gas. After purging with PBS, the mice were reflux-fixed in 4% paraformaldehyde phosphate buffer (Wako Pure Chemical Corporation), and the maxillary bone was removed. The maxillary bone was subsequently immersed overnight in PBS containing 20% sucrose. We prepared the frozen sections at a thickness of 20 µm from OCT blocks (Sakura Seiki, Tokyo, Japan) after they had been placed in a freezer at − 80 °C for 10 min; they were then in Leica CM3050 S (Leica, Nussloch, Germany) at − 20 °C for 10 min.

Hematoxylin–eosin (HE) staining

HE staining was performed using Meyer hematoxylin (Mutoh Chemical, Tokyo, Japan) and 1% eosin Y solution (Mutoh Chemical). Tissue specimens were sealed in ProLong™ Glass Antifade Mountant (Thermo Fisher Scientific) and observed under an optical microscope.

Immunostaining

The morphology of the periodontal ligament nerve fibers was observed using the ABC method. Sections were washed with PBS and treated with methanol (Wako Pure Chemical Corporation) containing 0.003% hydrogen peroxide (Wako Pure Chemical Corporation) for 30 min to inactivate endogenous peroxidase, and then washed with PBS and stained with rabbit polyclonal anti-CGRP (1:5000; Sigma-Aldrich, #C8198) and rabbit polyclonal anti-RAMP1 (1:5000; Abcam, #ab203282) and reacted for 16–18 h. Thereafter, they are washed with PBS, reacted with biotinylated swine anti-rabbit IgG (1:500; Dako, Copenhagen, Denmark) for 90 min, washed with PBS, and reacted with ABC complex (Vector Laboratories, Burlingame, CA, USA) for 90 min. After washing with PBS, ABC complex (Vector Laboratories, Burlingame, CA, USA) was reacted for 90 min. The HRP was subsequently visualized in 0.05 M Tris–HCl buffered saline, pH 7.6, containing 0.04% 3,3-diaminobenzidine (Sigma-Aldrich) and 0.003% hydrogen peroxide solution6 and sensitized with 0.1% nickel ammonium sulfate (Wako Pure Chemical Corporation). All the reactions were performed at room temperature. Tissue specimens were air-dried after the reactions, dehydrated in an ascending ethanol series, permeabilized with G-NOX (Genostaff, Tokyo, Japan), encapsulated in Multi Mount 480 (Matsunami Glass Industries, Osaka, Japan), and observed under an optical microscope.

Statistical analysis

Experimental data are presented as mean ± standard error. Significant difference tests were performed using Student’s-t test for two-group comparisons and Tukey’s test as post-hoc following analysis of variance (ANOVA) for multi-group comparisons. P < 0.05 was considered a significant difference.

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