Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

Summary

This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.

Abstract

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial process of alveolar bone remodeling in response to mechanical force, but the underlying mechanism remains elusive. Previous studies have been unable to reveal the precise mechanism of bone remodeling in any time and space due to animal model-related restrictions. The signal transducer and activator of transcription 3 (STAT3) is important in bone metabolism, but its role in osteoblasts during OTM is unclear. To provide in vivo evidence that STAT3 participates in OTM at specific time points and in particular cells during OTM, we generated a tamoxifen-inducible osteoblast lineage-specific Stat3 knockout mouse model, applied orthodontic force, and analyzed the alveolar bone phenotype.

Micro-computed tomography (Micro-CT) and stereo microscopy were used to access OTM distance. Histological analysis selected the area located within three roots of the first molar (M1) in the cross-section of the maxillary bone as the region of interest (ROI) to evaluate the metabolic activity of osteoblasts and osteoclasts, indicating the effect of orthodontic force on alveolar bone. In short, we provide a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding new light on skeletal mechanical biology.

Introduction

It is generally known that bone is under constant reconstruction throughout life, in response to mechanical forces according to Wolff's law^1,2. Appropriate mechanical stimulation, such as gravity and daily exercise, maintains bone mass and strength and prevents bone loss by stimulating both osteoblasts and osteoclasts. Osteoclasts, responsible for bone resorption^3,4,5,6,7, and osteoblasts, responsible for bone formation^8,9,10, maintain bone homeostasis and function jointly in the biological process of bone remodeling. In contrast, in the absence of loading stimuli, as in astronauts under long-term microgravity, bones suffer 10% bone mineral density loss, thus increasing the risk of osteoporosis^11,12. Furthermore, noninvasive and convenient mechanical therapies, including orthodontics and distraction osteogenesis, have emerged as treatments for bone diseases^13,14. All these have shown that mechanical force plays a critical role in maintaining bone quality and quantity. Recent studies generally analyzed bone remodeling in response to mechanical loading using time-consuming models such as running wheel and tail suspension tests, which usually took 4 weeks or more to simulate force loading or unloading^15,16. Therefore, there is demand for a convenient and efficient animal model for studying bone remodeling driven by force loading.

The alveolar bone is the most active in terms of bone remodeling, with a high turnover rate¹⁷. Orthodontic tooth movement (OTM), a common treatment for malocclusion, is an artificial process of alveolar bone remodeling in response to mechanical force. However, OTM, which induces rapid bone remodeling¹⁸, is also a time-saving way to study the effects of mechanical force on bone remodeling compared with other models with a long experimental period. Therefore, OTM is an ideal model to study bone remodeling under mechanical stimuli. It is noteworthy that the mechanism of alveolar bone remodeling is often time-sensitive, and it is necessary to observe the changes in alveolar bone remodeling at certain time points after modeling. With the dual advantages of temporal and spatial control of DNA recombination and tissue specificity, an inducible conditional gene knockout mouse model is a suitable choice for OTM studies.

Conventionally, OTM-mediated alveolar bone remodeling has been divided into tension zones involving bone formation and pressure zones involving bone resorption^19,20,21, which is more detailed but difficult to regulate. Furthermore, Yuri et al. reported that the time of bone formation in OTM differed on the tension and compression sides²². In addition, a previous study had demonstrated that the first molar could initiate wide remodeling of the maxillary alveolar bone under orthodontic force, which was not constrained to the tension and pressure zones²³. Therefore, we selected the area located within three roots of M1 in the cross-section of the maxillary bone as the region of interest (ROI) and described methods to assess the activity of osteoblasts and osteoclasts in the same area to evaluate alveolar bone remodeling under OTM.

As a nuclear transcription factor, signal transducer and activator of transcription 3 (STAT3) has been proven critical in bone homeostasis^24,25. Previous studies have reported low bone mineral density and recurrent pathological fractures in Stat3-mutant mice^26,27. Our previous study demonstrated that deletion of Stat3 in Osx⁺ osteoblasts caused craniofacial malformation and osteoporosis, as well as spontaneous bone fracture²⁸. Recently, we provided in vivo evidence with an inducible osteoblast-specific Stat3 deletion mouse model (Col1α2^CreERT2; Stat3^fl/fl, hereafter called Stat3^Col1α2ERT2) that STAT3 is critical in mediating the effects of orthodontic force driving alveolar bone remodeling²⁹. In this study, we provide methods and protocols for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding light on skeletal mechanical biology.

Protocol

All methods involving animals described here were approved by the ethics committee of the Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine (no. 82101048).

1. Establishing inducible osteoblast lineage-specific Stat3 knockout mice

NOTE: Stat3^fl/fl mice were obtained commercially; the Col1α2^CreERT2 strain was a gift (see the Table of Materials for all details). Standardized laboratory pellet food and water and standard laboratory environmental conditions (room temperature at 22 °C to 26 °C and humidity at 50%-55%) were provided for all the animals.

1. Put one sexually mature male mouse with two female mice in the same cage. After 18 days, check for newborns every day. Remove any pregnant female mice to an empty cage and keep them alone if necessary. Put male mice into other different breeding cages if the female mice are not pregnant within 30 days.
2. Generate Stat3^fl/+; Col1α2^CreERT2 mice by hybridizing Stat3^{fl/ fl} mice with Col1α2^CreERT2 mice; maintain all these mice on the C57BL/6 background. Collect 2-5 mm tail tips for genotyping and keep the male Stat3^fl/+; Col1α2^CreERT2 mice until they are sexually mature (F1).
3. Hybridize 6-week-old male Stat3^{fl/ +}; Col1α2^CreERT2 mice with female Stat3^fl/fl mice. Collect 2-5 mm tail tips when mice are 2 weeks old for genotyping, and keep the male Stat3^{fl/ fl}; Col1α2^CreERT2(Stat3^Col1α2ERT2) mice until they are sexually mature (F2).
4. Hybridize 6-week-old male Stat3^{fl/ fl}; Col1α2^CreERT2 mice with female Stat3^fl/fl mice (F3). Collect 2-5 mm tail tips when mice are 2 weeks old for genotyping, and use the younger mice of the same genotype to replace older breeding mice when appropriate (F3+N).
   NOTE: The reproductive capacity of mice decreases after age 8 months, so the mice in the breeding cages must be carefully monitored and replaced as needed. In addition, according to the experimental requirements, the number of breeding cages should be appropriately increased to avoid the extinction of the target gene mice.

2. Inducible deletion of Stat3 in Col1α2-expressing osteoblasts by tamoxifen

1. Dissolve tamoxifen in corn oil to 20 mg/mL in a centrifuge tube, and protect from light by wrapping in foil. Put the foil-covered centrifuge tube on a rotary mixer and mix at room temperature until completely dissolved.
   NOTE: Tamoxifen was obtained commercially and stored in the dark at 4 °C.
2. Select ten 6-week-old male mice and divide them into Stat3^fl/fl and Stat3^Col1α2ERT2 groups with five mice in each group. Administer tamoxifen (100 mg·kg^-1·bw) every 2 days for 1 week by intraperitoneal injection.

3. Orthodontic tooth movement (OTM) model

1. Prepare a sterilized plastic mouse dissection platform as an operating table to immobilize the mice.
   NOTE: The mouse dissection table was obtained commercially and consisted of four adjustable rubber posts and one metal rod for immobilizing the mice. Use sterile instruments and tools throughout the procedure.
2. Attach an elastic band to each rubber post for limb fixation.
3. Tie one elastic band between the two upper rubber posts; tie a thread to the elastic band to hold the lower incisors; and tie another thread to the metal rod to hold the upper incisors.
4. Anesthetize 7-week-old male mice with 0.1 mL solution of dexmedetomidine hydrochloride (0.1 mg·kg^-1·bw) and zoletil (tiletamine hydrochloride for 20 mg·kg^-1·bw and zolazepam hydrochloride for 20 mg·kg^-1·bw) dissolved in saline by intraperitoneal injection prior to surgery. Make sure that the mice are under proper anesthesia throughout the operation.
   NOTE: Pay attention to the effective use date of drugs, and do not use expired drugs. Zoletil exhibits potent analgesic and surgical anesthesia properties.
5. Confirm that the mice are under proper anesthesia by squeezing the toes of the hind limbs with fingers.
   NOTE: If the mice do not respond to the test, it means they are unconscious and the anesthesia has reached the desired depth. At that time, the mice are in a state of limb muscle relaxation, with smooth breathing and heart rate, and the operation can be started.
6. Use vet ointment on the eyes of the mice to prevent dryness and place the anesthetized mouse in the supine position on the operating table. Use four elastic bands on the rubber posts to fix the limbs, one thread attached to the metal rod to hold the upper incisors and another one attached to an elastic band between the two upper rubber posts to hook over the lower incisors to hold the lower jaw open.
7. Prepare closed-coil springs (0.25 mm wire size, 0.76 mm diameter, 1 mm length) for orthodontic force appliance.
   NOTE: Cut eight threads of spring for each mouse. Four threads of 1 mm in length in the middle for force appliance and two threads on each end for ligaturing using steel ligature wire.
8. Ligate one end of the spring prepared to the maxillary left first molar. Ligate the other end to the central incisor with a 0.1 mm steel ligature wire reinforced by light-curing restorative resin after applying adhesive to the incisors with the Q-tip to generate a stable force of a magnitude of 10 g measured by using a dynamometer.
9. After completing the operation, put the mice in a cage with others of the same strain in recovery, or put each mouse into an empty cage alone. Make sure the mice are not left unattended until they have regained sufficient consciousness 2−4 h after the operation to maintain sternal recumbency. For the next few days, feed the mice a soft diet and observe them regularly to ensure that no complications occur and to ascertain the degree of postsurgical pain; administer analgesic drugs as needed.
   ​NOTE: Mice that have undergone surgery should not be returned to the company of other mice until fully recovered. Do not put postsurgical mice in recovery with mice that are not anesthetized. Mice must be kept warm during recovery.
10. Check the orthodontic appliance every day and exclude any experimental mice with dislodgement.

4. Specimen collection

1. Euthanize the mice at three different time points via cervical dislocation: 4 days (d4), 7 days (d7), and 10 days (d10) after the start of OTM.
2. Use ophthalmic scissors to cut off the skin vertically from the cervical region and then separate the head from the body with the entire skin of the head dissected.
3. Cut off the skin and buccinator muscles from the bilateral angulus oris to the posterior region of the mandible. Completely disconnect the buccal muscles and the tendons attached to the coracoids to remove the mandible and trim extra bones to obtain complete maxillae. Then, remove the bone behind the bilateral third molars and tear off the palatal mucosa. Finally, disconnect the orthodontic appliance and cut off the bone between the incisors along the median palatine suture to obtain the alveolar bone of the right and left sides.
   ​NOTE: Ensure that the region from the incisors to the third molar is kept intact on both sides.

5. Preparation for paraffin section

1. Fixation: Immerse the harvested alveolar bone in 4% paraformaldehyde for 48 h and trim with ophthalmic scissors in the fume hood for sections 6 and 7.
2. Decalcification: Gently wash specimens with 1x PBS for 3 x 10 min. Decalcify the specimens in universal tissue fixative (pH 8.0), replaced with fresh solution every 2 days, for 5 weeks until the bones can be easily penetrated by a needle tip.
3. Dehydration: Wash specimens in 1x PBS for 3 x 10 min, and then sequentially immerse them in 95% ethanol, 100% ethanol, and xylene, for 2 x 1 h each solution.
4. Immerse specimens in a 1:1 mix of xylene and paraffin for 30 min and then in paraffin at 65 °C overnight.
5. Embedding: Select suitable embedding tanks. Place the alveolar bone uniformly with teeth up at the same level. Remove the specimens from the embedding tank and transfer them to a -20 °C freezer, then number them when the paraffin is fully cooled and solid.
6. Using a microtome, cut twenty to forty 4 µm-thick sections continuously in the transverse plane and float on 37 °C water. Adhere the sections to microscope slides and bake at 42 °C overnight.

6. OTM distance measurement

1. Photograph specimens from three time points vertically from the occlusal plane by stereo microscopy.
2. Scan the alveolar bone with a Micro-CT scanner. Reconstruct 3D images of the maximum sagittal plane of the maxillary alveolar bone, in which three molars are completely visible, using the scanner's supporting software following the manufacturer's instructions.
3. Measure the OTM distance using ImageJ software:
   1. Open ImageJ software and use the Straight line tool to create a line segment of known distance.
   2. Click Analyze and choose Set Scale to enter the value of the drawn line distance in Known distance and enter units in Unit of length.
   3. Draw a line between the midpoints of the mesial marginal ridge of M2 and the distal marginal ridge of M1 and click Measurement. The results shown in the Length column represent the OTM distance.

7. Histological analysis

1. Region of Interest (ROI) selection: Define the alveolar bone of the first molar (M1) as the ROI, located exactly within three roots of M1 in the transverse section at the maxillary bone. This region not only reaches the cortical bone of the first molar in the buccal-lingual direction, but also extends to the middle of the long axis of the mesiobuccal root, including the half area between the distobuccal root and the palatal root.
2. Analysis of osteogenesis
   1. Calcein and alizarin red double labeling and analysis
      1. Calcein and alizarin red preparation: Dissolve calcein in 2% NaHCO₃ solution to 1 mg/mL and alizarin red S in H₂O to 2 mg/mL.
         NOTE: In this study, calcein and alizarin red were obtained commercially.
      2. Administer calcein (20 mg·kg^-1·bw) on day 1 and alizarin red S (40 mg·kg^-1·bw) on day 8 by intraperitoneal injection after the commencement of OTM. Euthanize the mice on day 10 and harvest the alveolar bone.
      3. Prepare specimens and embed them following steps 5.1 and 5.3-5.5. For more details, refer to Yang et al.³⁰.
      4. Using a rotary microtome, cut 5 µm-thick sections continuously in the transverse plane. Adhere the sections to microscope slides and mount with coverslips using neutral balsam.
         NOTE: The rest of the samples must be stored with desiccant at room temperature.
      5. Examine and photograph the sections under a fluorescence microscope and calculate the mineral apposition rate (MAR) and bone formation rate (BFR/BS) according to the method previously described³⁰.
   2. Immunofluorescence
      1. Select suitable paraffin sections and bake at 65 °C for 30 min.
      2. Dewaxing: Immerse the sections in xylene for 5 min and repeat twice with fresh xylene each time.
      3. Rehydration: Immerse the sections sequentially in 95% ethanol, 75% ethanol, 50% ethanol, and ddH₂O, each for 5 min.
      4. Gently wash the sections with 1 x PBS for 2 x 5 min.
      5. Antigen retrieval: Immerse the sections in a mixture of Tris-HCl (0.05 M, pH 8.0), EDTA (0.01 M, pH 8.0), and protease K (10 µg/mL) in ddH₂O and incubate at 37 °C for 15 min.
      6. Gently wash the sections with 1 x PBS for 3 x 5 min.
      7. Block: Remove extra fluid from the sections and draw a circle around the target area using a hydrophobic marker. To block nonspecific binding, cover with a blocking buffer containing 10% bovine serum albumin and incubate at room temperature for 1 h.
         NOTE: Be careful not to dry the sections for too long, so as not to affect the results.
      8. Primary antibody incubation: Dilute the anti-osteopontin (OPN) antibody with antibody diluent to the recommended concentration, and add 30-50 µL to each sample; incubate at 4 °C overnight in a humidified chamber.
         NOTE: Be sure to leave some water in the chamber and cover it to prevent the antibody fluid from evaporating.
      9. Gently wash the sections with 1 x PBS for 3 x 10 min.
         NOTE: Steps 7.2.2.10-7.2.2.12 should be performed in the dark.
      10. Fluorescent secondary antibody incubation: Select an appropriate fluorescent secondary antibody corresponding to the primary antibody and dilute it to the recommended concentration. Add 30-50 µL to each sample and incubate at room temperature for 1 h.
      11. Gently wash the sections with 1 x PBS for 2 x 10 min.
      12. Use an antifade mounting medium with 4'6-diamidino-2-phenylindole (DAPI) to mount the specimens. Store the sections in the dark and photograph them as soon as possible.
      13. Carry out fluorescence microscopy incorporating a digital camera to examine and photograph the sections and count positive cells in the regions of interest (ROIs).
3. Analysis of osteoclastogenesis
   1. Tartrate-resistant acid phosphatase (TRAP) staining
      1. Select suitable paraffin sections. Dewax and rehydrate following steps 7.2.2.1-7.2.2.3.
      2. Prepare fresh staining solution using the TRAP staining kit according to the manufacturer's instructions and preheat to 37°C.
      3. Add 30-50 µL of staining solution to each sample and incubate at 37 °C in a humidified chamber for 20-30 min. Check every 5 min until red multinucleated osteoclasts are seen under a light microscope. Stop the reaction with ddH2O.
      4. Counterstain in hematoxylin solution for 30 s and immerse in 1% ammonia solution for 1 min for stable blue color. Rinse under slow-running tap water.
      5. Mount the sections with coverslips using neutral balsam and dry overnight.
      6. Capture photographs under a microscope and count the number of TRAP-positive cells with more than three nuclei, following our previous protocol³⁰.
   2. Immunofluorescence: Use cathepsin K (CTSK) antibody at the recommended concentration for immunofluorescence and follow the protocol described in section 7 above.

Results

Using this protocol, we established an inducible osteoblast lineage-specific Stat3 knockout mouse (Stat3^Col1α2ERT2) model to examine the effects of STAT3 deletion on orthodontic force-driven alveolar bone remodeling (Figure 1A,B). STAT3 deletion in osteoblasts was confirmed by immunofluorescence staining of alveolar bone (Figure 1C).

Stereo microscopy indicated that the OTM distance o...

Discussion

As malocclusion is among the most common oral disorders impairing breathing, mastication, speaking, and even appearance, the demand for orthodontics is increasing day by day with the incidence rising from 70% to 93% according to a previous epidemiological survey^31,32. How to accelerate alveolar bone remodeling to raise the efficiency of orthodontic treatment safely has become a hot topic in this field; therefore, it is necessary to clarify the mechanism of alveol...

Materials

Name	Company	Catalog Number	Comments
1x PBS	Beijing Solarbio Science & Technology Co.,Ltd.	P1020
4% paraformaldehyde	Wuhan Servicebio Technology Co., Ltd.	G1101
Alizarin red	Sigma-Aldrich	A5533
Anti-CTSK antibody	Santa Cruz	sc-48353
Anti-OPN antibody	R&D Systems, Minneapolis, MN, USA	AF808
Calcein	Sigma-Aldrich	C0875
Closed-coil springs	Innovative Material and Devices, Shanghai, China	CS1006B
Col1α2CreERT2 mice			A gift from Bin Zhou, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Dexmedetomidine hydrochloride	Orionintie Corporation, Orion Pharma Espoo site
EDTA	Beyotime Biotechanology	ST069
Embedding tanks	Citotest Labware Manufacturing Co., Ltd	80106-1100-16
Ethanol	Sinopharm Chemical Reagent Co., Ltd.	100092183
ImageJ software	NIH, Bethesda, MD, USA
Mounting medium with DAPI	Beyotime Biotechanology	P0131
Mouse dissection platform	Shanghai Huake Experimental Devices and Materials Co., Ltd.	HK105
Paraffin	Sangon biotech Co., Ltd.	A601889
Primers for genotyping			Stat3 F-TTGACCTGTGCTCCTACAAAAA; Stat3 R-CCCTAGATTAGGCCAGCACA; Cre F-CGATGCAACGAGTGATGAGG; Cre R-CGCATA ACCAGTGAAACAGC
Protease K	Sigma-Aldrich	539480
Self-curing restorative resin	3M ESPE, St. Paul, MN, USA	712-035
Stat3fl/fl mice	GemPharmatech Co., Ltd	D000527
Tamoxifen	Sigma-Aldrich	T5648
TRAP staining kit	Sigma-Aldrich	387A
Tris-HCl	Beyotime Biotechanology	ST780
Universal tissue fixative	Wuhan Servicebio Technology Co., Ltd.	G1105
Xylene	Sinopharm Chemical Reagent Co., Ltd.	10023418
Zoletil	VIRBAC