Name: Author Spotlight: Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential
Uploaded: 2023-07-21T12:20:00
Description: Watch this Scientific Journal Video about Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential at JoVE.com

This work was supported in part by grants from the National Natural Science Foundation of China (81870740, 82071083, 82271006, 82101048, 81800949); the Natural Science Foundation of Shanghai (21ZR1436900, 22ZR1436700); the Program of Shanghai Academic/Technology Research Leader (20XD1422300); Clinical Research Plan of SHDC (SHDC2020CR4084); the Cross-disciplinary Research Fund of Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine (JYJC201902, JYJC202116); the Innovation Research Team of High-Level Local Universities in Shanghai (SSMUZLCX20180501); the Research Discipline Fund no. KQYJXK2020 from Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine, and College of Stomatology, Shanghai Jiao Tong University; Original Exploration Project of Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine (JYYC003); Two-Hundred Talent Project of Shanghai Jiao Tong University School of Medicine; the Biomaterials and Regenerative Medicine Institute Cooperative Research Project Shanghai Jiao Tong University School of Medicine (2022LHB02); the Project of Biobank of Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine (YBKB201909, YBKB202216).

All methods involving animals described here were approved by the ethics committee of the Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine (no. 82101048).
1. Establishing inducible osteoblast lineage-specific Stat3 knockout mice
NOTE: Stat3fl/fl&#160;mice were obtained commercially; the Col1&#945;2CreERT2 strain was a gift (see the Table of Materials</strong...

Analyzing Alveolar Bone Remodeling to Explore Skeletal Mechanical Biology

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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 survey31,32. 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...

It is generally known that bone is under constant reconstruction throughout life, in response to mechanical forces according to Wolff&#39;s law1,2. 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 resorption3,4,5,6,7, and osteoblasts, responsible for bone formation8,9,10, 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 osteoporosis11,12. Furthermore, noninvasive and convenient mechanical therapies, including orthodontics and distraction osteogenesis, have emerged as treatments for bone diseases13,14. 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 unloading15,16. 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 resorption19,20,21, 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 homeostasis24,25. Previous studies have reported low bone mineral density and recurrent pathological fractures in Stat3-mutant mice26,27. 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 (Col1&#945;2CreERT2; Stat3fl/fl, hereafter called Stat3Col1&#945;2ERT2) 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.

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	<tbody>
		<tr>
			<td>1x PBS</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd.&#160;</td>
			<td>P1020</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Wuhan Servicebio Technology Co., Ltd.</td>
			<td>G1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CTSK antibody</td>
			<td>Santa Cruz</td>
			<td>sc-48353</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-OPN antibody</td>
			<td>R&#38;D Systems, Minneapolis, MN, USA</td>
			<td>AF808</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Closed-coil springs</td>
			<td>Innovative Material and Devices, Shanghai, China</td>
			<td>CS1006B</td>
			<td></td>
		</tr>
		<tr>
			<td>Col1&#945;2CreERT2 mice</td>
			<td></td>
			<td></td>
			<td>A gift from Bin Zhou, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.</td>
		</tr>
		<tr>
			<td>Dexmedetomidine hydrochloride</td>
			<td>Orionintie Corporation, Orion Pharma Espoo site</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Beyotime Biotechanology</td>
			<td>ST069</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding tanks</td>
			<td>Citotest Labware Manufacturing Co., Ltd</td>
			<td>80106-1100-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>100092183</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH, Bethesda, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium with DAPI</td>
			<td>Beyotime Biotechanology</td>
			<td>P0131</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse dissection platform</td>
			<td>Shanghai Huake Experimental Devices and Materials Co., Ltd.</td>
			<td>HK105</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sangon biotech Co., Ltd.</td>
			<td>A601889</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers for genotyping</td>
			<td></td>
			<td></td>
			<td>Stat3 F-TTGACCTGTGCTCCTACAAAAA; Stat3 R-CCCTAGATTAGGCCAGCACA; Cre F-CGATGCAACGAGTGATGAGG; Cre R-CGCATA ACCAGTGAAACAGC</td>
		</tr>
		<tr>
			<td>Protease K</td>
			<td>Sigma-Aldrich</td>
			<td>539480</td>
			<td></td>
		</tr>
		<tr>
			<td>Self-curing restorative resin</td>
			<td>3M ESPE, St. Paul, MN, USA</td>
			<td>712-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Stat3fl/fl mice</td>
			<td>GemPharmatech Co., Ltd</td>
			<td>D000527</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP staining kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Beyotime Biotechanology</td>
			<td>ST780</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal tissue fixative</td>
			<td>Wuhan Servicebio Technology Co., Ltd.</td>
			<td>G1105</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sinopharm Chemical Reagent Co., Ltd.</td>
			<td>10023418</td>
			<td></td>
		</tr>
		<tr>
			<td>Zoletil</td>
			<td>VIRBAC&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

using inducible osteoblastic lineage-specific stat3 knockout mice to study alveolar bone remodeling during orthodontic tooth movement

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.

Author Spotlight: Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential 

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

Our research focuses on the mechanism of bone remodeling under mechanical force, including alveolar bone and long bones. We try to answer how bones respond to mechanical stimuli to shed new light on skeletal mechanical biology for further clinical treatment. Currently, studies on OTM were limited to gene expression analysis in vivo or cellular function analysis in vitro.To this end, a challenge arose. How to trace and explore the function of specific genes in specific lineages during OTM in vivo? That's why we use inducible conditional knockout mice here.Orthodontic tooth movement, which induces rampant bone remodeling, is a time-saving method to study the effects of mechanical force on bone remodeling compared to other models that have a long experimental period. Our protocol also reveals dynamic alveolar bone remodeling at different time points with inducible gene-specific knockout mice. There are many important cell lineages in the alveolar bone.We aim to trace them and find out their underlying mechanisms during OTM. Furthermore, we will compare the mechanisms between alveolar bone and long bone remodeling to provide further clues for the potential of OTM model in biomechanical research.

Using this protocol, we established an inducible osteoblast lineage-specific Stat3 knockout mouse (Stat3Col1&#945;2ERT2) 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...

Watch this Scientific Journal Video about Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential at JoVE.com

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.

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 ...

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Research

JoVE Journal

Medicine

Murine Orthodontic Tooth Movement (OTM) Model

Begin by selecting 10 six-week old male mice and placing them into two groups labeled WT and CKO, with five mice in each group. Administer Tamoxifen to all the mice every two days for a week. Prepare a plastic mouse dissection platform as an operating table to immobilize the mice.Attach an elastic band to each rubber post for limb fixation. Tie another thread to the metal rod to hold the upper incisors. Place the anesthetized mouse in the supine position on the operating table.Use four elastic bands to fix the limbs. One thread attached to the metal rod to hold the upper incisors. Next, prepare closed coil springs for orthodontic force application.Ligate one end of the prepared spring to the maxillary left first molar. Ensure a stable force of 10 grams by measuring with the dynamometer. Then ligate the other end to the central incisor using a 0.1 millimeter resin reinforced steel ligature wire.After the operation, place the mice in a recovery cage. Monitor the mice for two to four hours until they regain sufficient consciousness to maintain sternal recumbency. Provide a soft diet and observe the mice regularly for complications, administering analgesic drugs as needed.

Murine Alveolar Bone Specimen Collection 

Begin by cutting the skin vertically off the cervical region of the euthanized mice separate the head from the body, with the entire skin of the head dissected. Next, 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 tendons from the coracoids to remove the mandible.Trim any extra bones to obtain the complete maxillae. Remove the bone behind the bilateral third molars, and peel off the palatal mucosa. Finally, disconnect the orthodontic appliance, and cut the bone between the incisors along the median palatine suture to obtain the right and left alveolar bone.Alveolar bone remodeling in the STAT3 deletion in osteoblasts was confirmed by immunofluorescent staining of alveolar bone. Stereomicroscopy revealed that the orthodontic tooth movement distance of the wild type mice increased on days four, seven, and 10. However, the orthodontic tooth movement distance was reduced in the STAT3 knockout mice.Micro-CT analysis on day 10 confirmed the reduced movement, indicating that STAT3 deletion decelerated orthodontic tooth movement.