Identification of the expression of SDF-1 in this animal model was analyzed alongside the SDF-1 and RAP relationship in this model through the localized injection of anti-SDF-1 neutralizing antibody, therefore, divided the analysis methods into two distinctive parts. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Tokyo Medical and Dental University (A2021-115C) and were performed in accordance with relevant guidelines and regulations. Our study is reported in accordance with ARRIVE guidelines.
Animals
Six-week-old male Wistar/ST rats, shipped at 5-week-old, with a mean weight of 180 g, were used in the study. All animals were housed in the same room under controlled temperature, humidity, and light conditions. A standard alternating 12-h light/dark environment was maintained. All animals were given a week for acclimatization, a powdered diet (RI-Sterile feed CE-2 powder type; CLEA Japan, Tokyo, Japan), and water ad libitum. The powdered food diet was replaced on the day of the experiment (tooth extraction, OTM, and medicine injection) to reduce trauma to the soft tissue after tooth extraction, ease pain, and reduce the risk of orthodontic appliance damage. The health status and body weight of the rats were evaluated on the first three days after the surgical procedures and every other day throughout the experiment. All procedures were performed under general anesthesia.
Tooth extraction and orthodontic tooth movement
Sixty-three rats were randomly allocated to three groups of 21 rats each.
- 1.
PBS Control group: M1 tooth extraction, M2 OTM, PBS injection
- 2.
IgG Control group: M1 tooth extraction, M2 OTM, IgG isotype control antibody injection (10 μg/0.1 mL)
- 3.
SDF-1 Antibody group: M1 tooth extraction, M2 OTM, anti-SDF-1 neutralizing monoclonal antibody injection (10 μg/0.1 mL)
All treatment procedures were performed following a subcutaneous injection of three-mixed anesthesia (medetomidine, 0.3 mg/kg; midazolam, 4 mg/kg; butorphanol, 5 mg/kg). The rats were weighed to calculate the anesthetic drug dose prior to each injection. To generate the RAP response, the left maxillary first molar was gently elevated and extracted with the least possible damage to the adjacent second molar. Subsequently, OTM was initiated using a 10-g force nickel-titanium alloy closed-coil tension spring (Tomy International, Tokyo, Japan) attached to the cervical ligature wire loop (Tomy International; diameter: 0.20 mm) around the left maxillary second molar to the ligature wire loop at the most cervical portion of the upper incisors and fixed with light-cured composite resin (GC, Tokyo, Japan). This generated a constant 10 g mesial force after activation, as measured by a force gauge (Supplementary Fig. 1a–c).
After the coil spring placement, the experimental group was given a local injection of anti-SDF-1 neutralizing monoclonal antibody (MAB310; R&D Systems, Minneapolis, MN, USA) at a volume and concentration of 10 μg/0.1 mL. Injections were administered in the buccal and palatal gingival mucosa of the interproximal area between the left maxillary first and second molar, 0.05 mL on each side. The control group received either a PBS injection or IgG isotype control antibody (MAB002; R&D Systems) injection at the same volume, concentration, and injection site as the experimental group. The contralateral side of the maxilla was not treated in all groups. All rats were treated with penicillin (Benzathine G penicillin, 20,000 IU) and painkiller (Buprenorphine, 0.01 mg/mL) intramuscularly after the surgical procedures. Medetomidine antagonists were administered for recovery from general anesthesia.
Appliances were routinely checked and cleaned on the first three days and every other day to prevent any possible mechanical damage to the coil spring and loosening of the wire ligatures.
Seven rats from each group were sacrificed 1, 3, and 7 days after the surgical procedures, according to their timeline. Serum samples were collected from all rats to quantify the SDF-1 protein concentration in the peripheral blood using an ELISA (n = 7). Tissues were evaluated for bone morphology, histological changes (n = 3), and molecular biology by quantitative reverse transcription PCR (RT-qPCR) (n = 4) (Supplementary Fig. 1d).
With the objective of identifying SDF-1 expression in this model, we observed the relationship between SDF-1 and the RAP via the localized injection of an anti-SDF-1 neutralizing antibody. The analyses of the obtained results were separated into two parts.
Part 1. To test the SDF-1expression, the untreated control side and the M1 extraction and M2 OTM experimental side of the control PBS injection group were used. Using all three time point subgroups, the collected maxillae were halved, and the two sides within the same group were compared. Analysis of the presence and location of SDF-1 was performed by IF staining. In addition, PCR analysis was performed to confirm the mRNA expressions of SDF-1 and its receptor CXCR4 at the transcriptional level.
Part 2. To assess the effects of SDF-1 blockade using a neutralizing antibody on the extraction of M1 and OTM in the M2 model, all groups and subgroups were compared using only their experimental sides. Micro-computed tomography (micro-CT) analysis of tooth movement and bone morphology, ELISA of blood serum to test the systemic effects of the medicine, histological analysis using hematoxylin and eosin (H&E) staining, histochemical staining for TRAP, IHC staining, IF staining, and RT-qPCR were performed to quantify gene expression levels.
Micro-CT analysis
In vivo micro-CT (R_mCT2 SPMD; Rigaku, Tokyo, Japan) of the rat head was performed both before and immediately after the surgical procedures for all rats. Preoperative micro-CT data were used as a baseline for M2 tooth movement measurements, and post-operative micro-CT data were used to check for broken root remnants of M1 immediately after tooth extraction. During the active tooth movement period, micro-CT imaging was performed under general anesthesia on days 1, 3, and 7 of the experimental timeline for each group. All imaging conditions were set at 90 kV, 160 mA, and a 30 mm field of view. The tooth movement distance of M2 was measured from the micro-CT images using two methods.
- 1.
Distance from the distal surface of M2 to the mesial surface of maxillary third molar (M3).
- 2.
The measurement from the reference line was created from the opposite M3 of the maxillary right untreated side to the distal surface of M2.
The mesial movement of M3 was also analyzed using the second method (Supplementary Fig. 2a–c).
Three rats from each subgroup were randomly selected after sacrifice for histological and bone morphological analyses. Dissected maxillae were immediately fixed by immersing in 4% paraformaldehyde (pH 7.4, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 4 °C for 48 h and stored in PBS pH 7.4 at 4 °C. Samples were scanned using a micro-CT system coupled to a desktop X-ray micro-CT system (SMX-100CT; Shimadzu, Kyoto, Japan) with output settings of 75 kV and 140 mA, and a scanning resolution of 9.5 μm. The ROI for structural morphometry analysis was the interradicular alveolar bone of the maxillary M2, defined by the borders of the septum between the roots of the M2. Using trabecular bone analysis software (TRI/3D-BON; Ratoc, Tokyo, Japan), bone parameters regarding BV/TV, BMD, Tb.Th, Tb.N, and Tb.Sp were analyzed in the selected ROI. Evaluations of the micro-CT data were repeated at least three times by a single examiner to calculate the average values (Supplementary Fig. 2d,e).
Enzyme-linked immunosorbent assay
Serum SDF-1 protein levels were determined using an ELISA kit (MCX120; R&D Systems), following the manufacturer’s protocol. Under general anesthesia, 5 mL of blood was drawn via cardiac puncture at the time of sacrifice. Serums were separately collected using a serum separator blood collecting tube (BD Vacutainer® SST™ II Plus; BD, Franklin Lakes, NJ, USA), centrifuged at 1000 g for 10 min, and stored at − 80 °C until analysis. The ELISA was performed in duplicate for each sample. The colorimetric density of the developed microplate was determined using a Multiskan Sky Microplate Spectrophotometer and SkanIt software (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. Wavelength corrections were performed by subtracting the readings at 540 nm or 570 nm from those at 450 nm for optical imperfection correction.
Hematoxylin and eosin staining
Following micro-CT imaging, the specimens were decalcified in 10% disodium ethylenediamine tetraacetate in phosphate buffer at pH 7.4 for six weeks, dehydrated in a series of ascending ethanol concentrations, cleared with xylene, infiltrated, and embedded in paraffin. A rotary microtome was used to obtain 4 μm-thick serial sections in a horizontal plane through the first, second, and third-molar roots. The sections were mounted on coated glass slides, deparaffinized with xylene, and stained with H&E. Morphological and histological changes were examined by light microscopy (DXm1200; Nikon, Tokyo, Japan) using NIS-Elements D Imaging Software (Version 2.30, Nikon).
Immunofluorescence staining
The expression of SDF-1 and its presence and location were evaluated by IF staining using rabbit anti-SDF-1/CXCL12 polyclonal antibody (Bioss, Woburn, MA, USA). Deparaffinized and rehydrated sections were blocked with 5% bovine serum albumin for 60 min at room temperature (25 °C). Sections were incubated with primary antibody (rabbit anti-SDF-1/CXCL12 polyclonal antibody; Bioss) (1:100 dilution) overnight at 4 °C. Secondary antibodies (Donkey Anti-Rabbit IgG H&L Alexa Fluor® 594; Abcam, Cambridge, UK) (1:200 dilution) were used to incubate the sections for 40 min in room temperature. Quenching of autofluorescence (Vector TrueVIEW; Vector Labs, Burlingame, CA, USA) was performed to remove unwanted fluorescence in the tissue sections following the manufacturer’s protocol. Sections were mounted with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Labs, Burlingame, CA, USA), as provided in the kit. Fluorescent images were evaluated within 48 h of mounting using a BZ-X710 fluorescence microscope (Keyence, Itasca, IL, USA).
Tartrate-resistant acid phosphatase staining
To analyze catabolic activity in the alveolar bone, multinucleated osteoclasts and preosteoclasts were stained with tartrate-resistant acid phosphatase (TRAP). After deparaffinization and rehydration, the sections were stained using a TRAP staining kit (Wako Pure Chemical, Osaka, Japan) according to the manufacturer’s protocol. Incubation of the sections in TRAP buffer for 30 min was performed, followed by washing with distilled water, and counterstained with 0.02% Fast Green (Wako Pure Chemical, Osaka, Japan) nuclear staining solution for 10 min. The number of TRAP-positive multinucleated osteoclasts in the PDL area around the M2 mesial roots was counted by a single examiner in three randomized sections for each sample and the averages were calculated.
Immunohistochemistry staining
The sections were stained with the following primary antibodies for the IHC analyses: IL-1β, IL-6, RANKL, and Cathepsin K (Bioss, Woburn, MA, USA) (1:400 dilution). After deparaffinization and rehydration, sections were treated with 3% hydrogen peroxide (Abcam) for 10 min to quench the endogenous peroxidase activity, followed by the incubation with normal goat serum to block the non-specific binding for 30 min at room temperature. Primary antibodies with different specific concentrations were applied to the sections and incubated overnight at 4 °C. The following day, the slides were incubated with the biotinylated secondary antibody for 30 min using the VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Labs, Burlingame, CA, USA). Subsequently, the prepared VECTASTAIN ABC reagent was applied to the slides and incubated for another 30 min. Sections were stained with 3,3’-Diaminobenzidine (DAB) (Abcam) and counterstained with hematoxylin.
Quantitative reverse transcription PCR
Four rats from each subgroup were randomly selected for the quantification of gene expression using RT-qPCR. Immediately after sacrifice, the maxillae were dissected from the skulls and any attached tissues were removed. The maxilla was trimmed with sterile scissors and the M2 clinical crown was removed using a sterile orthodontic wire cutter. The attached gingival tissue was discarded, leaving the final sample consisting only of the M1 extraction socket, M2 roots with the adjacent PDL attached, and the surrounding bone at a distance of approximately 1–2 mm. For the untreated negative control, tissue samples were collected as described in Part 1. The M1 crown was removed along with the M2 crown and the surrounding tissue sample size was identical to that previously stated. Tissue samples were transferred to liquid nitrogen and snap-frozen immediately after collection. A sterile mortar and pestle with liquid nitrogen was used to grind the collected samples into small pieces. The crushed samples were further homogenized, and total RNA was isolated using a TRIzol® reagent (Invitrogen; Thermo Fisher Scientific). Complimentary DNA was synthesized from total RNA with reverse transcription using PrimeScript™ RT Master Mix (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Real-time PCR analysis was done in triplicate for each sample using Probe qPCR Mix (Takara Bio) and commercially obtained rat’s specific primers for CXCL12/SDF-1, CXCR4, IL-1β, RANKL, OPG and GAPDH (TaqMan Gene Expression Assay, Applied Biosystems; Thermo Fisher Scientific). Gene expression levels were calculated using the comparative Ct method and normalized to those of GAPDH.
