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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

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

Published: July 21st, 2023

DOI:

10.3791/65613

1Center of Craniofacial Orthodontics, Department of Oral and Cranio-maxillofacial Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 2College of Stomatology, Shanghai Jiao Tong University, 3National Center for Stomatology, 4National Clinical Research Center for Oral Diseases, 5Shanghai Key Laboratory of Stomatology, 6Shanghai Starriver Bilingual School, 7The 2nd Dental Center, Ninth People’s Hospital, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine
* These authors contributed equally

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 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.

It is generally known that bone is under constant reconstruction throughout life, in response to mechanical forces according to Wolff'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<....

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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: Stat3fl/fl mice were obtained commercially; the Col1α2CreERT2 strain was a gift (see the Table of Materials

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Using this protocol, we established an inducible osteoblast lineage-specific Stat3 knockout mouse (Stat3Col1α2ERT2) 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.......

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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.......

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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'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's Hospital, Shanghai....

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NameCompanyCatalog NumberComments
1x PBSBeijing Solarbio Science & Technology Co.,Ltd. P1020
4% paraformaldehydeWuhan Servicebio Technology Co., Ltd.G1101
Alizarin redSigma-AldrichA5533
Anti-CTSK antibodySanta Cruzsc-48353
Anti-OPN antibodyR&D Systems, Minneapolis, MN, USAAF808
CalceinSigma-AldrichC0875
Closed-coil springsInnovative Material and Devices, Shanghai, ChinaCS1006B
Col1α2CreERT2 miceA gift from Bin Zhou, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Dexmedetomidine hydrochlorideOrionintie Corporation, Orion Pharma Espoo site
EDTABeyotime BiotechanologyST069
Embedding tanksCitotest Labware Manufacturing Co., Ltd80106-1100-16
EthanolSinopharm Chemical Reagent Co., Ltd.100092183
ImageJ softwareNIH, Bethesda, MD, USA
Mounting medium with DAPIBeyotime BiotechanologyP0131
Mouse dissection platformShanghai Huake Experimental Devices and Materials Co., Ltd.HK105
ParaffinSangon biotech Co., Ltd.A601889
Primers for genotypingStat3 F-TTGACCTGTGCTCCTACAAAAA; Stat3 R-CCCTAGATTAGGCCAGCACA; Cre F-CGATGCAACGAGTGATGAGG; Cre R-CGCATA ACCAGTGAAACAGC
Protease KSigma-Aldrich539480
Self-curing restorative resin3M ESPE, St. Paul, MN, USA712-035
Stat3fl/fl miceGemPharmatech Co., LtdD000527
TamoxifenSigma-AldrichT5648
TRAP staining kitSigma-Aldrich387A
Tris-HClBeyotime BiotechanologyST780
Universal tissue fixativeWuhan Servicebio Technology Co., Ltd.G1105
XyleneSinopharm Chemical Reagent Co., Ltd.10023418
ZoletilVIRBAC 

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