Name: using inducible osteoblastic lineage-specific stat3 knockout mice to study alveolar bone remodeling during orthodontic tooth movement
Uploaded: 2023-07-21T12:20:00.000+00:00
Duration: 5 min 25 s
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 for all details). Standardized laboratory pellet food and water and standard laboratory environmental conditions (room temperature at 22 &#176;C to 26 &#176;C and humidity at 50%-55%) were provided for all the animals.
<ol>
	<li>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.</li>
	<li>Generate Stat3fl/+; Col1&#945;2CreERT2 mice by hybridizing Stat3fl/ fl mice with Col1&#945;2CreERT2 mice; maintain all these mice on the C57BL/6 background. Collect 2-5 mm tail tips for genotyping and keep the male Stat3fl/+; Col1&#945;2CreERT2 mice until they are sexually mature (F1).</li>
	<li>Hybridize 6-week-old male Stat3fl/ +; Col1&#945;2CreERT2 mice with female Stat3fl/fl mice. Collect 2-5 mm tail tips when mice are 2 weeks old for genotyping, and keep the male Stat3fl/ fl; Col1&#945;2CreERT2(Stat3Col1&#945;2ERT2) mice until they are sexually mature (F2).</li>
	<li>Hybridize 6-week-old male Stat3fl/ fl; Col1&#945;2CreERT2 mice with female Stat3fl/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.</li>
</ol>
2. Inducible deletion of Stat3 in Col1&#945;2-expressing osteoblasts by tamoxifen
<ol>
	<li>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:&#160;Tamoxifen was obtained commercially and stored in the dark at 4 &#176;C.</li>
	<li>Select ten 6-week-old male mice and divide them into Stat3fl/fl and Stat3Col1&#945;2ERT2 groups with five mice in each group. Administer tamoxifen (100 mg&#183;kg-1&#183;bw) every 2 days for 1 week by intraperitoneal injection.</li>
</ol>
3. Orthodontic tooth movement (OTM) model
<ol>
	<li>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.</li>
	<li>Attach an elastic band to each rubber post for limb fixation.</li>
	<li>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.</li>
	<li>Anesthetize 7-week-old male mice with 0.1 mL solution of dexmedetomidine hydrochloride (0.1 mg&#183;kg-1&#183;bw) and zoletil (tiletamine hydrochloride for 20 mg&#183;kg-1&#183;bw and zolazepam hydrochloride for 20 mg&#183;kg-1&#183;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#8722;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. 
	&#8203;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.</li>
	<li>Check the orthodontic appliance every day and exclude any experimental mice with dislodgement.</li>
</ol>
4. Specimen collection
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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. 
	&#8203;NOTE: Ensure that the region from the incisors to the third molar is kept intact on both sides.</li>
</ol>
5. Preparation for paraffin section
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Immerse specimens in a 1:1 mix of xylene and paraffin for 30 min and then in paraffin at 65 &#176;C overnight.</li>
	<li>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 &#176;C freezer, then number them when the paraffin is fully cooled and solid.</li>
	<li>Using a microtome, cut twenty to forty 4 &#181;m-thick sections continuously in the transverse plane and float on 37 &#176;C water. Adhere the sections to microscope slides and bake at 42 &#176;C overnight.</li>
</ol>
6. OTM distance measurement
<ol>
	<li>Photograph specimens from three time points vertically from the occlusal plane by stereo microscopy.</li>
	<li>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&#39;s supporting software following the manufacturer&#39;s instructions.</li>
	<li>Measure the OTM distance using ImageJ software:
	<ol>
		<li>Open ImageJ software and use the Straight line tool to create a line segment of known distance.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
7. Histological analysis
<ol>
	<li>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.</li>
	<li>Analysis of osteogenesis
	<ol>
		<li>Calcein and alizarin red double labeling and analysis
		<ol>
			<li>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.</li>
			<li>Administer calcein (20 mg&#183;kg-1&#183;bw) on day 1 and alizarin red S (40 mg&#183;kg-1&#183;bw) on day 8 by intraperitoneal injection after the commencement of OTM. Euthanize the mice on day 10 and harvest the alveolar bone.</li>
			<li>Prepare specimens and embed them following steps 5.1 and 5.3-5.5. For more details, refer to Yang et al.30.</li>
			<li>Using a rotary microtome, cut 5 &#181;m-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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Immunofluorescence
		<ol>
			<li>Select suitable paraffin sections and bake at 65 &#176;C for 30 min.</li>
			<li>Dewaxing: Immerse the sections in xylene for 5 min and repeat twice with fresh xylene each time.</li>
			<li>Rehydration: Immerse the sections sequentially in 95% ethanol, 75% ethanol, 50% ethanol, and ddH2O, each for 5 min.</li>
			<li>Gently wash the sections with 1 x PBS for 2 x 5 min.</li>
			<li>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 &#181;g/mL) in ddH2O and incubate at 37 &#176;C for 15 min.</li>
			<li>Gently wash the sections with 1 x PBS for 3 x 5 min.</li>
			<li>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.</li>
			<li>Primary antibody incubation: Dilute the anti-osteopontin (OPN) antibody with antibody diluent to the recommended concentration, and add 30-50 &#181;L to each sample; incubate at 4 &#176;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.</li>
			<li>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.</li>
			<li>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 &#181;L to each sample and incubate at room temperature for 1 h.</li>
			<li>Gently wash the sections with 1 x PBS for 2 x 10 min.</li>
			<li>Use an antifade mounting medium with 4&#39;6-diamidino-2-phenylindole (DAPI) to mount the specimens. Store the sections in the dark and photograph them as soon as possible.</li>
			<li>Carry out fluorescence microscopy incorporating a digital camera to examine and photograph the sections and count positive cells in the regions of interest (ROIs).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Analysis of osteoclastogenesis
	<ol>
		<li>Tartrate-resistant acid phosphatase (TRAP) staining
		<ol>
			<li>Select suitable paraffin sections. Dewax and rehydrate following steps 7.2.2.1-7.2.2.3.</li>
			<li>Prepare fresh staining solution using the TRAP staining kit according to the manufacturer&#39;s instructions and preheat to 37&#176;C.</li>
			<li>Add 30-50 &#181;L of staining solution to each sample and incubate at 37 &#176;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.</li>
			<li>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.</li>
			<li>Mount the sections with coverslips using neutral balsam and dry overnight.</li>
			<li>Capture photographs under a microscope and count the number of TRAP-positive cells with more than three nuclei, following our previous protocol30.</li>
		</ol>
		</li>
		<li>Immunofluorescence: Use cathepsin K (CTSK) antibody at the recommended concentration for immunofluorescence and follow the protocol described in section 7 above.</li>
	</ol>
	</li>
</ol>

Analyzing Alveolar Bone Remodeling to Explore Skeletal Mechanical Biology

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+STAT3+is+crucial+for+orthodontic+force+driving+alveolar+bone+remodeling+and+tooth+movement.">Osteoblastic STAT3 is crucial for orthodontic force driving alveolar bone remodeling and tooth movement.</a> Journal of Bone and Mineral Research. 38 (1), 214-227 (2023).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+phenotype+analysis+of+a+conditional+Stat3+deletion+mouse+model.">Skeletal phenotype analysis of a conditional Stat3 deletion mouse model.</a> Journal of Visualized Experiments: JoVE. (161), 61390 (2020).</li><li>Egic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+orthodontic+malocclusion+in+schoolchildren+in+Slovenia.+A+prospective+aepidemiological+study.">Prevalence of orthodontic malocclusion in schoolchildren in Slovenia. A prospective aepidemiological study.</a> European Journal of Paediatric Dentistry. 23 (1), 39-43 (2022).</li><li>Gois, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+malocclusion+between+primary+and+mixed+dentitions+among+Brazilian+children+A+5-year+longitudinal+study.">Incidence of malocclusion between primary and mixed dentitions among Brazilian children A 5-year longitudinal study.</a> The Angle Orthodontist. 82 (3), 495-500 (2012).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+Of+triptolide+on+tooth+movement+and+root+resorption+in+rats.">Effects Of triptolide on tooth movement and root resorption in rats.</a> Drug Design, Development and Therapy. 13, 3963-3975 (2019).</li><li>Wang, C., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+gene+knockout+mice.">Progress in gene knockout mice.</a> Sheng Wu Gong Cheng Xue Bao. 35 (5), 784-794 (2019).</li><li>Cao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-induced+Adrb2+in+periodontal+ligament+cells+promotes+tooth+movement.">Force-induced Adrb2 in periodontal ligament cells promotes tooth movement.</a> Journal of Dental Research. 93 (11), 1163-1169 (2014).</li></ol>

The authors have no conflicts of interest to declare.

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, Stat3Col1&#945;2ERT2, 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 Stat3Col1&#945;2ERT2 and wild-type mice and nothing special in breeding. We have previously found that the inducible deletion of Stat3 in Col1&#945;2+ osteoblasts impaired bone formation and reduced bone mass in adult mice28. In this study, the bone metabolism level of Stat3Col1&#945;2ERT2 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.

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.

<table><tbody><tr><td>1x PBS</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.&amp;nbsp;</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&amp;amp;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>&lt;em&gt;Col1&amp;alpha;2&lt;sup&gt;CreERT2&lt;/sup&gt;&lt;/em&gt; 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>&lt;em&gt;Stat3&lt;sup&gt;fl/fl&lt;/sup&gt; &lt;/em&gt;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&amp;nbsp;</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.

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 of WT mice increased on d4, d7, and d10. However, in Stat3Col1&#945;2ERT2 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 Stat3Col1&#945;2ERT2 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 Stat3Col1&#945;2ERT2 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 Stat3Col1&#945;2ERT2 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65613/65613fig01.jpg" /> 
Figure 1: Illustration of inducible osteoblast lineage-specific Stat3 knockout mouse model generation. (A) Schematic diagram of Stat3 knockout in Col1&#945;2-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&#183;kg-1&#183;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 &#181;m. This figure is from Gong et al. 26. Abbreviations: STAT3 = signal transducer and activator of transcription 3; Col1&#945; = collagen 1-alpha; TA = tamoxifen; WT = wild type; OTM = orthodontic tooth movement; OPN = osteopontin; DAPI = 4&#39;,6-diamidino-2-phenylindole. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65613/65613fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65613/65613fig02.jpg" /> 
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 Stat3Col1&#945;2ERT2 mice. (C) Representative images of non-OTM sides and OTM sides in WT and Stat3Col1&#945;2ERT2 mice on d10 after OTM by micro-CT. n = 5. Scale bars = 1 mm (B), 500 &#181;m (C). This figure is from Gong et al.26. Abbreviations: STAT3 = signal transducer and activator of transcription 3; Col1&#945; = collagen 1-alpha; WT = wild type; OTM = orthodontic tooth movement; SAG = sagittal plane. Dashed curves indicate the OTM distance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65613/65613fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65613/65613fig03.jpg" /> 
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 &#181;m (B), 100 &#181;m (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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65613/65613fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65613/65613fig04.jpg" /> 
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 Stat3Col1&#945;2ERT2 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 Stat3Col1&#945;2ERT2 mice. n = 5. (C) TRAP staining and quantification of TRAP-positive cells in alveolar bone on d4 after OTM in WT and Stat3Col1&#945;2ERT2 mice. n = 5. (D) Immunofluorescence staining and quantification of CTSK+ cells in alveolar bone on d4 after OTM in WT and Stat3Col1&#945;2ERT2 mice. n = 5. Scale bars = 10 &#181;m (A), 50 &#181;m (B-D). This figure is from Gong et al.26. Abbreviations: STAT3 = signal transducer and activator of transcription 3; Col1&#945; = collagen 1-alpha; WT = wild type; OTM = orthodontic tooth movement; OPN = osteopontin; DAPI = 4&#39;,6-diamidino-2-phenylindole; TRAP = tartrate-resistant acid phosphatase; MAR = mineral apposition rates; CTSK = cathepsin K. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65613/65613fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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.

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 ...

using-inducible-osteoblastic-lineage-specific-stat3-knockout-mice-to

Nanyang Technological University

Research

JoVE Journal

Medicine

1.3K Views.  Shanghai Jiao Tong University School of Medicine. Ons onderzoek richt zich op het mechanisme van botremodellering onder mechanische kracht, inclusief alveolair bot en lange botten. We proberen een antwoord te geven op de vraag hoe botten reageren op mechanische stimuli om een nieuw licht te werpen op de mechanische biologie van het skelet voor verdere klinische behandeling. Momenteel waren studies over OTM beperkt tot genexpressie-analyse in vivo of cellulaire functie-analyse in vitro.Daartoe ontstond een uitdaging. Hoe kan de functie...

Video: Auteur in de schijnwerpers: vergelijking van alveolaire en lange botremodellering om het potentieel van OTM-modellen te verkennen 

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Video Movement Analysis Using Smartphones (ViMAS): A Pilot Study

The use of smartphones in clinical practice is steadily increasing with the availability of low cost/freely available &#34;apps&#34; that could be used to assess human gait. The primary aim of this manuscript is to test the concurrent validity of kinematic measures recorded by a smartphone application in comparison to a 3D motion capture system in the sagittal plane. The secondary aim was to develop a protocol for clinicians on the set up of the smartphone camera for video movement analysis.
The sagittal plane knee angle was measured during heel strike and toe off events using the smart phone app and a 3D motion-capture system in 32 healthy subjects. Three trials were performed at near (2-m) and far (4-m) smartphone camera distances. The order of the distances was randomized. Regression analysis was performed to estimate the height of the camera based on either the subject&#39;s height or leg length.
Absolute measurement errors were least during toe off (3.12 &#177; 5.44 degrees) compared to heel strike (5.81 &#177; 5.26 degrees). There were significant (p &#60; 0.05) but moderate agreements between the application and 3D motion capture measures of knee angles. There were also no significant (p &#62; 0.05) differences between the absolute measurement errors between the two camera positions. The measurement errors averaged between 3 - 5 degrees during toe off and heel strike events of the gait cycle.
The use of smartphone apps can be a useful tool in the clinic for performing gait or human movement analysis. Further studies are needed to establish the accuracy in measuring movements of the upper extremity and trunk.

Video Movement Analysis Using Smartphones ViMAS: A Pilot Study

This manuscript describes the method to test the concurrent validity of kinematic measures recorded by the smartphone application in comparison to a 3D motion capture system in the sagittal plane. This protocol will enable clinicians to set up smartphones for video capture of human movement.

The use of smartphones in clinical practice is steadily increasing with the availability of low cost/freely available &#34;apps&#34; that could be used to ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to understand these complex remodeling processes, it is critical to study the tooth and periodontal tissues within their 3D context and therefore minimize any sectioning and tissue artefacts. Mouse models are often utilized in developmental and structural biology, as well as in biomechanics due to their small size, high metabolic rate, genetics and ease of handling. In principle this also makes them excellent models for dental related studies. However, a major impediment is their small tooth size, the molars in particular. This paper is aimed at providing a step by step protocol for generating orthodontic tooth movement and two methods for 3D imaging of the periodontal ligament fibrous component of a mouse mandibular molar. The first method presented is based on a micro-CT setup enabling phase enhancement&#160;imaging of fresh collagen tissues. The second method is a bone clearing method using ethyl cinnamate that enables imaging through the bone without sectioning and preserves endogenous fluorescence. Combining this clearing method with reporter mice like Flk1-Cre;TdTomato provided a first of its kind opportunity to image the 3D vasculature in the PDL and alveolar bone.

We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning.

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to ...

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms and pharmaceutical treatments of osteoporosis. Cathepsin K (Ctsk)-Cre mice have been widely used for functional studies of osteoclasts. The signal transducer and activator of transcription 3 (STAT3) is relevant in bone homeostasis, but its role in osteoclasts in vivo remains poorly defined. To provide the in vivo evidence that STAT3 participates in osteoclast differentiation and bone metabolism, we generated an osteoclast-specific Stat3 deletion mouse model (Stat3 fl/fl; Ctsk-Cre) and analyzed its skeletal phenotype. Micro-CT scanning and 3D reconstruction implied increased bone mass in the conditional knockout mice. H&#38;E staining, calcein and alizarin red double staining, and tartrate-resistant acid phosphatase (TRAP) staining were performed to detect bone metabolism. In short, this protocol describes some canonical methods and techniques to analyze skeletal phenotype and to study the critical genes controlling osteoclast activity in vivo.

Skeletal Phenotype Analysis of a Conditional Stat3 Deletion Mouse Model

This protocol describes a canonical method to understand the critical genes controlling osteoclast activity in vivo. This method uses a transgenic mouse model and some canonical techniques to analyze skeletal phenotype.

Transgenic mouse models are powerful for understanding the critical genes controlling osteoclast differentiation and activity, and for studying mechanisms ...

Biology

Investigating Orthodontic Tooth Movement and Bone Adaptation Mechanisms in Mice

Studying Orthodontic Tooth Movement in Mice

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at tension sites, orchestrated by osteoclasts and osteoblasts, respectively. This mechanism serves as a valuable model for studying various aspects of bone adaptation, including root resorption and the cellular response to mechanical force stimuli. The protocol outlined here offers a straightforward approach to investigate OTM, establishing 0.35 N as the optimal force in a mouse model employing a nickel-titanium (NiTi) coil spring. Utilizing micro-computed tomography analysis, we quantified OTM by assessing the discrepancy in the linear distance at the cement-enamel junction. The evaluation also included an analysis of orthodontic-induced inflammatory root resorption, assessing parameters such as root mineral density and the percentage of root volume per total volume. This comprehensive protocol contributes to advancing our understanding of bone remodeling processes and enhancing the ability to develop effective orthodontic treatment strategies.

Here, we present a protocol for studying orthodontic tooth movement (OTM), serving as a suitable model for investigating the mechanisms of bone adaptation, root resorption, and the response of bone cells to mechanical stimuli. This comprehensive guide provides detailed information on the OTM model, micro-computed tomography acquisition, and subsequent analysis.

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at ...

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

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