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 formation8,9,10, 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 de 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 (Col1α2CreERT2; Stat3fl/fl, hereafter called Stat3Col1α2ERT2) 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.
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 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.
2. Inducible deletion of Stat3 de Col1α2-expressing osteoblasts by tamoxifen
3. Orthodontic tooth movement (OTM) model
4. Specimen collection
5. Preparation for paraffin section
6. OTM distance measurement
7. Histological analysis
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 of WT mice increased on d4, d7, and d10. However, in Stat3Col1α2ERT2 mice, OTM distance was reduced (Figure 2B). This phenomenon was also confirmed by Micro-CT analysis on d10, indicating that osteoblastic Stat3 deletion decelerated OTM (Figure 2C).
For histological analysis, the area located within three roots of M1 in the transverse section at the maxillary bone was defined as ROI, which shows the overall situation of alveolar bone remodeling (Figure 3A). The hematoxylin-eosin staining and OPN immunofluorescent staining showed the entire alveolar bone of ROI (Figure 3B,C).
For osteogenic analysis, alizarin red and calcein labeling indicated that orthodontic force increased the mineral apposition rate (MAR) of alveolar bone in WT mice, but the MAR in the OTM group of Stat3Col1α2ERT2 mice was reduced compared with the WT mice (Figure 4A). Furthermore, immunofluorescence staining demonstrated that the number of OPN+ osteoblasts increased under orthodontic force, but there were fewer OPN+ osteoblasts in the OTM group of Stat3Col1α2ERT2 mice than in the WT mice (Figure 4B). For osteoclastogenic analysis, TRAP staining indicated that, under orthodontic force, the number of osteoclasts increased in WT mice, but there were fewer osteoclasts in the OTM group of Stat3Col1α2ERT2 mice than in the WT mice (Figure 4C). This phenomenon was also confirmed by immunofluorescence staining of CTSK (Figure 4D). These results revealed that osteoblastic Stat3 deficiency reduced the activity of both osteoblasts and osteoclasts in response to orthodontic force and impaired bone remodeling during tooth movement.
Figure 1: Illustration of inducible osteoblast lineage-specific Stat3 knockout mouse model generation. (A) Schematic diagram of Stat3 knockout in Col1α2-expressing osteoblasts induced by tamoxifen (left). The scheme of the experiment: 6-week-old male wild-type and Stat3Col1ERT2 mice were administered TA (100 mg·kg-1·bw) every 2 days from a week before OTM model construction and sacrificed for analysis on d4, d7, and d10 after OTM (right). (B) Hybridization progress of inducible osteoblast lineage-specific Stat3 knockout mice. (C) Double immunofluorescence staining of STAT3 and OPN in the alveolar bone of WT and Stat3Col1ERT2 mice on d10 after OTM and the quantification of double-positive cells on non-OTM sides and OTM sides. n = 5; scale bars = 50 µm. This figure is from Gong et al. 26. Abbreviations: STAT3 = signal transducer and activator of transcription 3; Col1α = collagen 1-alpha; TA = tamoxifen; WT = wild type; OTM = orthodontic tooth movement; OPN = osteopontin; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 2: Osteoblastic deficiency of Stat3 decelerated orthodontic tooth movement (A) The schematic diagram of the OTM model in vivo. The spring ligated between the incisors and the first molar (M1) and induced orthodontic force to move M1 towards the mesial ridge. (B) The OTM distance of d0, d4, d7, and d10 after OTM in WT mice and Stat3Col1α2ERT2 mice. (C) Representative images of non-OTM sides and OTM sides in WT and Stat3Col1α2ERT2 mice on d10 after OTM by micro-CT. n = 5. Scale bars = 1 mm (B), 500 µm (C). This figure is from Gong et al.26. Abbreviations: STAT3 = signal transducer and activator of transcription 3; Col1α = collagen 1-alpha; WT = wild type; OTM = orthodontic tooth movement; SAG = sagittal plane. Dashed curves indicate the OTM distance. Please click here to view a larger version of this figure.
Figure 3: Illustration of regions of interest. (A) Schematic diagram of the regions of ROIs. The dotted area located within three roots of M1 in the cross-section of the maxillae was selected as ROI. The three roots of M1 include the mesiobuccal root, distobuccal root, and palatal root. (B) HE staining of the entire alveolar bone of M1 in mice. (C) Immunofluorescent staining of OPN in the entire alveolar bone of M1 in mice. Scale bars = 200 µm (B), 100 µm (C). This figure is from Gong et al.26. Abbreviations: ROIs = regions of interest; MB = mesiobuccal root; DB = distobuccal root; P = palatal root; HE = hematoxylin-eosin; OPN = osteopontin; PDL = periodontal ligament. Please click here to view a larger version of this figure.
Figure 4: Osteoblastic deficiency of Stat3 impaired orthodontic force-induced bone formation and resorption. (A) Sequential fluorochrome labeling by calcein and alizarin red S in WT and Stat3Col1α2ERT2 mice during OTM and quantification of mineral apposition rates. n = 5. (B) Immunofluorescence staining and quantification of OPN+ cells in alveolar bone on d10 after OTM in WT and Stat3Col1α2ERT2 mice. n = 5. (C) TRAP staining and quantification of TRAP-positive cells in alveolar bone on d4 after OTM in WT and Stat3Col1α2ERT2 mice. n = 5. (D) Immunofluorescence staining and quantification of CTSK+ cells in alveolar bone on d4 after OTM in WT and Stat3Col1α2ERT2 mice. n = 5. Scale bars = 10 µm (A), 50 µm (B–D). This figure is from Gong et al.26. Abbreviations: STAT3 = signal transducer and activator of transcription 3; Col1α = collagen 1-alpha; WT = wild type; OTM = orthodontic tooth movement; OPN = osteopontin; DAPI = 4',6-diamidino-2-phenylindole; TRAP = tartrate-resistant acid phosphatase; MAR = mineral apposition rates; CTSK = cathepsin K. Please click here to view a larger version of this figure.
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 alveolar bone remodeling driven by OTM. In previous studies, researchers used in vivo drug injection to study the mechanism of OTM in animal models21,33, which was simple and easy, but it was difficult to rule out the influence of drugs on the cells of other systems. Therefore, it is highly necessary to study the mechanism of bone remodeling under orthodontic force in vivo.
In recent years, gene knockout mice have been widely used, providing a solid foundation for the study of gene function and the exploration of therapeutic targets34. Among these techniques, whole gene knockout technology was the earliest approach. The Adrb2-/- adrenergic receptor (Adrb2) knockout mouse model was used to explore the role of the sympathetic nervous system in alveolar bone remodeling during OTM35. However, total gene-knockout mice may result in embryonic lethality in some cases, hindering the study of gene function. Therefore, conditional knockout mice were developed, targeting a gene in specific cell types. However, because research related to alveolar bone remodeling is often time-sensitive, it is, therefore, necessary to observe the changes in alveolar bone at specific time points after modeling.
Inducible conditional gene knockout mice have the dual advantages of temporal and spatial control of DNA recombination and tissue specificity, and gene knockout can be achieved at specific time points and specific tissues through the application of drugs or hormones, which can better meet the research needs. In addition, STAT3 is a transcription factor widely expressed in bone and other tissues and has the functions of regulating cell proliferation, differentiation, apoptosis, and immune and other important functions25. We previously reported that the bone mineral density of non-induced osteoblast-specific Stat3 knockout mice was reduced28, suggesting that non-induced conditional Stat3 knockout mice are not suitable for the mechanistic study of alveolar bone remodeling which needs to exclude the influence of development.
Therefore, in this study, we generated tamoxifen-inducible osteoblast-specific Stat3 gene knockout mice, Stat3Col1α2ERT2, by employing inducible conditional gene knockout technology based on the Cre/loxP system, which is controllable in time and space and more suitable for the study of the mechanism of alveolar bone remodeling, as well as allowing further study of the function of specific cells and genes combined with lineage tracing. Before the induction of tamoxifen, there was no obvious difference between Stat3Col1α2ERT2 and wild-type mice and nothing special in breeding. We have previously found that the inducible deletion of Stat3 de Col1α2+ osteoblasts impaired bone formation and reduced bone mass in adult mice28. In this study, the bone metabolism level of Stat3Col1α2ERT2 mice in non-OTM groups decreased, which was consistent with the previous results. Moreover, as the bone metabolism level of the OTM group decreased more significantly in response to orthodontic force, it could be considered that Stat3 played an important role in the regulation of bone metabolism under mechanical force. We further explored the mechanism of bone remodeling regulated by Stat3. The results indicated that Stat3 could directly promote osteoblast differentiation28,29 and affected osteoclast differentiation by regulating the crosstalk between osteoblasts and osteoclasts through the modulation of Mmp3 transcription29.
In this protocol, we selected the area located within three roots of M1 in the cross-section at the maxillary bone of mice as the ROI to evaluate alveolar bone remodeling. With this area, the analysis of alveolar bone is closer to that of long bones, in which the activity of osteogenesis and osteoclastogenesis are analyzed in the same region, rather than the traditional method of analyzing the characteristics of different sides. Furthermore, according to the classic hypothesis, two drugs with opposite effects should be applied on the pressure and tension sides, respectively, to accelerate the tooth movement rate. This method provided a theoretical basis for overcoming these bottlenecks. In this OTM model, the magnitude of orthodontic force was measured by using a dynamometer. However, the force would be slightly attenuated with the reduction in the distance between the first molar and incisors. Therefore, it is necessary to establish a standardized mechanical system to generate a more constant and stable orthodontic force.
Within the protocol, there are some critical steps that should be kept in mind. First, in the process of OTM model construction, the first molar and incisor should be firmly ligated to avoid the failure of force application by dislodgement. Second, attention must be paid to the direction of force, and horizontal force should be applied to the first molar to avoid tooth extraction caused by the application of vertical force. Finally, when embedding, the orientation of the specimen should be determined according to the target cross-section, and the sections should be observed in time to adjust the orientation to obtain ideal sections.
In conclusion, we provide protocols for an inducible, specific, gene knockout mouse model of OTM, which reveals alveolar bone remodeling at different time points. We will further use this to study the function of specific cells and genes combined with lineage tracing. We then set up a dynamic and cell-specific pattern in vivo in the field of OTM mechanism research, offering evidence for orthodontics in the clinic. In addition, this study provides detailed protocols for constructing an OTM model, which enables rapid bone remodeling by stimulating osteoblasts and osteoclasts and provides an ideal model for the study of bone remodeling in response to mechanical force. More than that, we describe methods for analyzing alveolar bone remodeling during OTM to provide a novel strategy for the study of skeletal mechanical biology.
The authors have nothing to disclose.
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 Jiao Tong University School of Medicine, and College of Stomatology, Shanghai Jiao Tong University; Original Exploration Project of Shanghai Ninth People'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's Hospital, Shanghai Jiao Tong University School of Medicine (YBKB201909, YBKB202216).
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 |