We thank for the laboratory platform and assistance of Ear Institute, Shanghai Jiaotong University School of Medicine. This work was supported by Shanghai Pujiang Program (22PJ1409200); National Natural Science Foundation of China (No.11932012); Postdoctoral Scientific Research Foundation of Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine;Fundamental research program funding of Ninth People&#39;s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (JYZZ154).

All experimental procedures described here were approved by the Animal Care Committee of Shanghai Ninth People&#39;s Hospital, Shanghai Jiao Tong University School of Medicine (SH9H-2023-A616-SB). 4-week-old C57BL/6 male mice were used in this study. All the instruments used were sterilized prior to the procedure.
1. Preparation of the suture expansion model
<ol>
	<li>Preparation of two retention holders.
	<ol>
		<li>Use 0.014&#39;&#39; Australian wire or stainless steel wir...

3D Visualization of Bone Remodeling Under Tensile Load in Suture Expansion Mice

<ol>
	<li>Opperman, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+sutures+as+intramembranous+bone+growth+sites.">Cranial sutures as intramembranous bone growth sites.</a> Developmental Dynamics. 219 (4), 472-485 (2000).</li><li>Thenier-Villa, J. L., Sanrom&#225;n-&#193;lvarez, P., Miranda-Lloret, P., Plaza Ram&#237;rez, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incomplete+reossification+after+craniosynostosis+surgery-incidence+and+analysis+of+risk+factors:+a+clinical-radiological+assessment+study.">Incomplete reossification after craniosynostosis surgery-incidence and analysis of risk factors: a clinical-radiological assessment study.</a> Journal Of Neurosurgery-pediatrics. 22 (2), 120-127 (2018).</li><li>Markiewicz, M. R., Recker, M. J., Reynolds, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+sagittal+and+lambdoid+craniosynostosis:+open+cranial+vault+expansion+and+remodeling.">Management of sagittal and lambdoid craniosynostosis: open cranial vault expansion and remodeling.</a> Oral And Maxillofacial Surgery Clinics Of North America. 34 (3), 395-419 (2022).</li><li>Mao, J. J., Wang, X., Kopher, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+craniofacial+sutures:+orthopedic+implications.">Biomechanics of craniofacial sutures: orthopedic implications.</a> Angle Orthodontist. 73 (2), 128-135 (2003).</li><li>Shayani, A., Sandoval Vidal, P., Garay Carrasco, I., Merino Gerlach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midpalatal+suture+maturation+method+for+the+assessment+of+maturation+before+maxillary+expansion:+a+systematic+review.">Midpalatal suture maturation method for the assessment of maturation before maxillary expansion: a systematic review.</a> Diagnostics (Basel). 12 (11), 2774 (2022).</li><li>Suzuki, 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=Miniscrew-assisted+rapid+palatal+expander+(MARPE):+the+quest+for+pure+orthopedic+movement.">Miniscrew-assisted rapid palatal expander (MARPE): the quest for pure orthopedic movement.</a> Dental Press Journal Of Orthodontics. 21 (4), 17-23 (2016).</li><li>Yu, M., 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=Cranial+suture+regeneration+mitigates+skull+and+neurocognitive+defects+in+craniosynostosis.">Cranial suture regeneration mitigates skull and neurocognitive defects in craniosynostosis.</a> Cell. 184 (1), 243-256 (2021).</li><li>Roth, D. M., Souter, K., Graf, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Craniofacial+sutures:+Signaling+centres+integrating+mechanosensation,+cell+signaling,+and+cell+differentiation.">Craniofacial sutures: Signaling centres integrating mechanosensation, cell signaling, and cell differentiation.</a> European Journal of Cell Biology. 101 (3), 151258 (2022).</li><li>Zhao, 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=The+suture+provides+a+niche+for+mesenchymal+stem+cells+of+craniofacial+bones.">The suture provides a niche for mesenchymal stem cells of craniofacial bones.</a> Nature Cell Biology. 17 (4), 386-396 (2015).</li><li>Jing, D., 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=Response+of+Gli1(+)+suture+stem+cells+to+mechanical+force+upon+suture+expansion.">Response of Gli1(+) suture stem cells to mechanical force upon suture expansion.</a> Journal of Bone And Mineral Research. 37 (7), 1307-1320 (2022).</li><li>Xu, Y., Malladi, P., Chiou, M., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+posterofrontal/sagittal+suture+mesenchymal+cells+in+vitro.">Isolation and characterization of posterofrontal/sagittal suture mesenchymal cells in vitro.</a> Plastic and Reconstructive Surgery. 119 (3), 819-829 (2007).</li><li>Kong, L., 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=Isolation+and+characterization+of+human+suture+mesenchymal+stem+cells+in+vitro.">Isolation and characterization of human suture mesenchymal stem cells in vitro.</a> International Journal of Stem Cells. 13 (3), 377-385 (2020).</li><li>Ikegame, M., 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=Tensile+stress+induces+bone+morphogenetic+protein+4+in+preosteoblastic+and+fibroblastic+cells,+which+later+differentiate+into+osteoblasts+leading+to+osteogenesis+in+the+mouse+calvariae+in+organ+culture.">Tensile stress induces bone morphogenetic protein 4 in preosteoblastic and fibroblastic cells, which later differentiate into osteoblasts leading to osteogenesis in the mouse calvariae in organ culture.</a> Journal of Bone And Mineral Research. 16 (1), 24-32 (2001).</li><li>Liu, S. S., Opperman, L. A., Buschang, P. H. <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+recombinant+human+bone+morphogenetic+protein-2+on+midsagittal+sutural+bone+formation+during+expansion.">Effects of recombinant human bone morphogenetic protein-2 on midsagittal sutural bone formation during expansion.</a> American Journal of Orthodontics And Dentofacialorthopedics. 136 (6), 768-769 (2009).</li><li>Liang, W., Ding, P., Li, G., Lu, E., Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyapatite+nanoparticles+facilitate+osteoblast+differentiation+and+bone+formation+within+sagittal+suture+during+expansion+in+rats.">Hydroxyapatite nanoparticles facilitate osteoblast differentiation and bone formation within sagittal suture during expansion in rats.</a> Drug Design Development and Therapy. 15, 905-917 (2021).</li><li>Morinobu, M., 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=Osteopontin+expression+in+osteoblasts+and+osteocytes+during+bone+formation+under+mechanical+stress+in+the+calvarial+suture+in+vivo.">Osteopontin expression in osteoblasts and osteocytes during bone formation under mechanical stress in the calvarial suture in vivo.</a> Journal of Bone And Mineral Research. 18 (9), 1706-1715 (2003).</li><li>Hwang, S., Chung, C. J., Choi, Y. J., Kim, T., Kim, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cetirizine,+a+histamine+1+receptor+antagonist,+on+bone+remodeling+after+calvarial+suture+expansion.">The effect of cetirizine, a histamine 1 receptor antagonist, on bone remodeling after calvarial suture expansion.</a> Korean Journal of Orthodontics. 50 (1), 42-51 (2020).</li><li>Jing, D., 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=Tissue+clearing+of+both+hard+and+soft+tissue+organs+with+the+PEGASOS+method.">Tissue clearing of both hard and soft tissue organs with the PEGASOS method.</a> Cell Research. 28 (8), 803-818 (2018).</li><li>Jing, D., 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=Tissue+clearing+and+its+application+to+bone+and+dental+tissues.">Tissue clearing and its application to bone and dental tissues.</a> Journal of Dental Research. 98 (6), 621-631 (2019).</li><li>Luo, W., 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=Investigation+of+postnatal+craniofacial+bone+development+with+tissue+clearing-based+three-dimensional+imaging.">Investigation of postnatal craniofacial bone development with tissue clearing-based three-dimensional imaging.</a> Stem Cells Development. 28 (19), 1310-1321 (2019).</li></ol>

We applied a standard suture expansion mouse model to observe the regular morphological changes that occur every week during the entire month-long remodeling cycle10. This model is useful for researching calvarial bone remodeling and regeneration by expanding calvarial sutures, as well as for studying various suture cells in vivo. To fully present the results of such research, three-dimensional visualization of stained tissues is needed. Therefore, PEGASOS technology, known for its effici...

Craniofacial sutures are fibrous tissues that connect craniofacial bones and play essential roles in the growth and remodeling of craniofacial bones. The structure of the suture resembles a river, providing a flow of cell resources to nourish and build the &#34;river bank&#34;, known as the osteogenic fronts, which contribute to the formation of craniofacial bones via intramembranous osteogenesis1.
Interest in craniofacial sutures has been driven by clinical needs to understand premature closure of cranial sutures and facial suture dysfunction, which may lead to craniofacial deformities and even life-threatening conditions in children. Open suturectomy is routinely used in clinical treatment, but long-term follow-up has shown incomplete re-ossification recurrence in some patients2. Minimally invasive craniotomy assisted by expansion springs or endoscopic stripe craniectomy may provide a safer approach to preserving the potential suture rather than discarding the tissues3. Similarly, orthopedic therapies such as facemasks and expansion appliances have been widely used to treat sagittal or horizontal maxillary hypoplasia, with some studies extending the age limitation to treat adult patients via miniscrew-assisted palatal expanders4,5,6. Additionally, cranial suture regeneration with mesenchymal stem cells (MSCs) combined with biodegradable materials is a potential therapy in the future, offering a novel direction for the treatment of related diseases7. However, the function process or regulatory mechanism of sutures remains elusive.
Bone remodeling mainly consists of a balance between bone formation conducted by osteoblasts and bone resorption conducted by osteoclasts, where osteogenic differentiation of stem cells stimulated by mechanical signals plays an important role. After decades of research, it has been found that craniofacial sutures are highly plastic mesenchymal stem cell niches8. Suture stem cells (SuSCs) are a heterogeneous group of stem cells, belonging to mesenchymal stem cells (MSCs) or bone stem cells (SSCs). SuSCs are labeled in vivo by four markers, including Gli1, Axin2, Prrx1, and Ctsk. Gli1+ SuSCs, in particular, have strictly verified the biological characteristics of stem cells, not only exhibiting high expression of typical MSC markers but also demonstrating excellent osteogenic and chondrogenic potential9. Previous research has shown that Gli1+ SuSCs actively contribute to new bone formation under tensile force, identifying them as the suture stem cell source supporting distraction osteogenesis10.
In the past, extensive mechanical characteristics of stem cells were studied in vitro via Flexcell, four-point bending, micro-magnet loading system, and others. Although mouse cranial suture-derived mesenchymal cells have been identified in vitro11, and human suture mesenchymal stem cells have also been isolated recently12, the biomechanical response of suture cells remains unclear in the in vitro system. To further investigate the bone remodeling process, a suture expansion model based on isolated calvaria organ culture has been established, paving the way for establishing a useful in vivo suture expansion model1,13. Rabbits14 and rats15 have been the most widely used animals in basic research for suture expansion. However, mice are preferred animal models for exploring human disease due to their highly homologous genome with humans, numerous gene modification lines, and strong reproductive hybridization ability. Existing mouse models of cranial suture expansion typically rely on stainless steel orthodontic spring wires to apply tensile force to the sagittal suture16,17. In these models, two holes are made in each side of the parietal bones to fix the expansion device, and the wires are embedded under the skin, which may affect the cell activation mode.
Regarding the visualization method, the two-dimensional observation of slices in the sagittal direction has been generally adopted for decades. However, considering that bone remodeling is a complex three-dimensional dynamic process, obtaining complete three-dimensional information has become an urgent need. The PEGASOS tissue transparency technique emerged to meet this requirement18,19. It offers unique advantages for the transparency of hard and soft tissues, enabling the complete bone remodeling process to be reproduced in three-dimensional space.
To gain a deeper and more comprehensive understanding of the physiological changes in the bone remodeling periods, a standard sagittal suture expansion mouse model with a spring setting between the handmade holders was established10. With a standardized acid etching and bonding procedure, the expansion device could be firmly bonded to the cranial bone, generating a tensile force perpendicular to the sagittal suture. Furthermore, the PEGASOS tissue clearing method was applied after double labeling of the mineralized bone post-expansion to fully visualize the bone modeling changes after suture expansion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>37% Acid etching</td>
			<td>Xihubiom</td>
			<td>E10-02/1807011</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A3882</td>
			<td></td>
		</tr>
		<tr>
			<td>AUSTRALIAN WIRE</td>
			<td>A.J.WILCOCK</td>
			<td>0.014&#39;&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B6630</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein green</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) sulfate, anhydrous</td>
			<td>Sangon Biotech</td>
			<td>A603008</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamometer</td>
			<td>Sanliang</td>
			<td>SF-10N</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU</td>
			<td>Invitrogen</td>
			<td>E104152</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Confocal Microscope</td>
			<td>Leica</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sangon Biotech</td>
			<td>E607008</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-MMA 500</td>
			<td>Sigma-Aldrich</td>
			<td>447943</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6148&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Meters</td>
			<td>Mettler Toledo</td>
			<td>S220</td>
			<td></td>
		</tr>
		<tr>
			<td>Quadrol</td>
			<td>Sigma-Aldrich</td>
			<td>122262</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sangon Biotech</td>
			<td>A500873</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sangon Biotech</td>
			<td>A610476</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring</td>
			<td>TAOBAO</td>
			<td>0.2*1.5*1*7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-Cyanine3 azide</td>
			<td>Lumiprobe</td>
			<td>A1330</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butanol</td>
			<td>Sigma-Aldrich</td>
			<td>360538&#160;</td>
			<td>Protect from light. Do not freeze.</td>
		</tr>
		<tr>
			<td>Transbond MIP 
			Moisture Insensitive Primer</td>
			<td>3M Unitek</td>
			<td>712-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Transbond XT 
			Light Cure Adhesive Paste</td>
			<td>3M Unitek</td>
			<td>712-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>V900257</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline</td>
			<td>Sangon Biotech</td>
			<td>A500027</td>
			<td></td>
		</tr>
	</tbody>
</table>

a 3-d visualization technique for bone remodeling in a suture expansion mouse model

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and facial bone growth, housing mesenchymal stem cells and osteoprogenitors. As most craniofacial bones develop through intramembranous ossification, the sutures' marginal regions act as initiation points. Due to this significance, these sutures have become intriguing targets in orthopedic therapies like spring-assisted cranial vault expansion, rapid maxillary expansion, and maxillary protraction. Under orthopedic tracing force, suture stem cells are rapidly activated, becoming a dynamic source for bone remodeling during expansion. Despite their importance, the physiological changes during bone remodeling periods remain poorly understood. Traditional sectioning methods, primarily in the sagittal direction, do not capture the comprehensive changes occurring throughout the entire suture. This study established a standard mouse model for sagittal suture expansion. To fully visualize bone remodeling changes post-suture expansion, the PEGASOS tissue clearing method was combined with whole-mount EdU staining and calcium chelating double labeling. This allowed the visualization of highly proliferating cells and new bone formation across the entire calvarial bones following expansion. This protocol offers a standardized suture expansion mouse model and a 3-D visualization method, shedding light on the mechanobiological changes in sutures and bone remodeling under tensile force loading.

Author Spotlight: PEGASOS Tissue Clearing Technique to Visualize Bone Remodeling 

A 3-D Visualization Technique for Bone Remodeling in a Suture Expansion Mouse Model

The interest in craniofacial sutures has been driven by the clinical needs to understand the mechanisms of orthodontic and the orthopedic treatment. The scope of our study is to establish a standardized suture expansion mouse model, and the 3D visualization method to study the mechanobiological changes of suture and bone remodeling upon expansion. PEGASOS tissue clearing technique has been combined with various labeling methods to obtain versatile information of suture and bond remodeling in space perspective.This model is suitable for various strengths of transgenic mice of different age. It is convenient to adjust the magnitude of tensile stress and withdraw force at any time. With the PEGASOS technique, we can observe the three dimensional spatial temporal distribution of cranial suture stem cells during bone remodeling.With the PEGASOS technique, we will further explore the relationship between suture stem cells and mechanical stress, as well as its specific mechanisms.

Using this protocol, a mouse model for sagittal suture expansion has been established (Figure 1-2). For 3-D visualization of bone modeling changes after suture expansion, the PEGASOS tissue clearing method was applied to the entire calvarial bones following expansion. After perfusion, calvarial bones were separated (Figure 3A), and the appropriate PEGASOS process was continued (Table 1 and Table 2</stron...

Watch this Scientific Journal Video about PEGASOS Tissue Clearing Technique to Visualize Bone Remodeling at JoVE.com

This protocol presents a standardized suture expansion mouse model and a 3-D visualization method to study the mechanobiological changes of the suture and bone remodeling under tensile force loading.

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and ...

a-3-d-visualization-technique-for-bone-remodeling-suture-expansion

Research

JoVE Journal

Biology

923 ビュー.  Shanghai Jiao Tong University School of Medicine. 頭蓋顔面縫合糸への関心は、歯列矯正と整形外科治療のメカニズムを理解したいという臨床的ニーズによって推進されてきました。本研究では、標準化された縫合糸拡張マウスモデルを確立し、3次元可視化法を用いて、縫合糸のメカノバイオロジカルな変化や拡張時の骨リモデリングを研究することを目指します。PEGASOS組織透明化技術は、宇宙の観点から縫合糸と結合...

ビデオ: 著者スポットライト:骨リモデリングを視覚化するためのPEGASOS組織透明化技術 

This protocol presents a standardized suture expansion mouse model and a 3-D visualization method to study the mechanobiological changes of the suture and bone remodeling under tensile force...

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

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...

Analyzing Alveolar Bone Remodeling to Explore Skeletal Mechanical Biology

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studying orthodontic tooth movement in mice

Author Spotlight: Analyzing Maxillary Bone Remodeling and Dental Root Resorption for Improved Dental Treatments

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...

Investigating Orthodontic Tooth Movement and Bone Adaptation Mechanisms in Mice

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less-invasive technique for non-stabilized mandibular fracture in mouse models

Author Spotlight: Novel Technique for Mandibular Endochondral Bone Repair with Minimal Tissue Damage

This protocol presents a comprehensive and efficient method for analyzing mandibular bone repair. We describe a reproducible technique for non-stabilized mandibular fracture in a mouse model, allowing analysis of the process of endochondral bone repair with minimal tissue damage and bone loss.

Standardized Mandibular Fracture Protocol for Bone Repair in Mice

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optical clearing and imaging of immunolabeled kidney tissue

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney...

a hydrophobic tissue clearing method for rat brain tissue

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both...

measuring bone remodeling and recreating the tumor-bone microenvironment using calvaria co-culture and histomorphometry

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its...

a tissue clearing method for neuronal imaging from mesoscopic to microscopic scales

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic...

the establishment of a murine maxillary orthodontic model

Author Spotlight: Insights into an Efficient Murine Maxillary Orthodontic Model Protocol

Here we demonstrate step-by-step a manageable, orthodontic tooth movement protocol operated on a murine maxillary model. With the explicit explanation of each step and visual demonstration, researchers can master this model and apply it to their experimental needs with a few...

A Murine Maxillary Orthodontic Model Guide to Bone Remodeling Analysis

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pegasos method to prepare transparent tissues and calvarial bones

PEGASOS Method to Prepare Transparent Tissues and Calvarial Bones

zebrafish scale regeneration in toto and ex vivo culture of scales

Author Spotlight: Use of Fish Scales for Bone Remodeling Research &amp;#8211; Advancements in Ex Vivo Imaging and Dissecting Cell Interactions

This protocol describes the harvesting and visualization of elasmoid scales of zebrafish during in vivo regeneration. In addition, the ex vivo culture of these scales for up to 7 days after harvest is...

Harvesting and Visualization of Elasmoid Scales of Zebrafish During In Vivo Regeneration

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ovarian tissue culture to visualize phenomena in mouse ovary

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian...

scanning electron microscopy of macerated tissue to visualize the extracellular matrix

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac...

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

exploring adipose tissue structure by methylsalicylate clearing and 3d imaging

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent...

colonoscopic guided pinch biopsy in a mouse model: a technique to visualize and extract tissue samples from mouse colon

This video demonstrates the protocol for performing colonoscope-guided pinch biopsy in the mouse. This technique helps visually analyze the colon lumen and collect tissue samples for detailed...

Colonoscopic Guided Pinch Biopsy in a Mouse Model: A Technique to Visualize and Extract Tissue Samples from Mouse Colon

adipo-clear: a tissue clearing method for three-dimensional imaging of adipose tissue

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for...

practical considerations for the design, execution, and interpretation of studies involving whole-bone bending tests of rodent bones

Author Spotlight: Enhancing Accuracy and Reproducibility in Whole Bone Bending Tests 

Mechanical testing of rodent bones is a valuable method for extracting information regarding a bone's susceptibility to fracture. Lacking proper practical understanding, the results may be overinterpreted or lack validity. This protocol will serve as a guide to ensure mechanical tests are performed accurately to provide valid and functional...

Principles and Protocols of Mechanical Testing for Bone Fracture Susceptibility

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Sagittal Suture Expansion Surgery to Prepare Mouse Suture Expansion Model

Begin by preparing two retention holders. Use light wire pliers and a 0.014 inch Australian wire to make a helical loop. Then prepare customized stainless steel springs and cut the spring into an appropriate length.Also prepare one straight Australian wire or straight steel wire of length seven millimeters and cut two pieces of paper with diameters of two millimeters to use as barriers. Next, place an anesthetized mouse on the operating table and carefully remove the fur on top of the head with hair removal cream or a shaver. Then disinfect the scalp of the mouse using alternating rounds of Iodoform and 75%alcohol.Position the mouse with its head on the top and use surgical tapes to fix the limbs on the operating table. Now, use surgical scissors to perform an arcuate flap along the scalp near the neck of the mouse, fully exposing the sagittal suture and surrounding skull. Then, fix the scalp flap with a 6-0 suture on the operating table.Dry the skull and etch it with 37%phosphoric acid for 20 seconds. Then use normal saline to clean up the acid etching. Place the two holders on both sides of the parietal bones, three millimeters from the sagittal sutures and glue them with a light cured adhesive.Then put back the scalp flap. Label the position of the holders and cut two small holes in the scalp at the corresponding positions. Reset the flap scalp by simultaneously passing the small loops through the holes to expose the skin surface.To install the spring and guide wire, cut the spring one millimeter longer than the distance between the two holders. Compress the pressure spring and place it between the small coils on both sides. Pass the stainless steel wire through the small coils and the spring and release the spring to obtain a starting thrust of about 30G.Next, place two scraps of paper between the spring and the holders and bond them with a light cured adhesive to set up barriers at both ends of the spring. After expansion, micro CT was used to visualize the changes in the cranial suture. In comparison to controls, the cranial suture gradually and significantly expanded after applying force for one day and fluffy bony protrusions appeared on the bone edge after day five.Also, visualization of mineralization using double labeling method, showed that force loading significantly activated osteogenesis. Newly mineralized bone was observed after seven days, indicating the bone remodeling process after suture expansion.

To begin, fix the limbs of an anesthetized mouse with adhesive tape. Hold the skin of the abdomen upward and carefully cut along the circumference of the thorax to fully expose it. Make a small cut in the right atrium with ophthalmic scissors and immediately insert a perfusion 22 gauge needle at the apex of the left ventricle.Then push 30 to 50 milliliters of cardiac perfusion fluid through the syringe. After the outflow fluid from the right atrium becomes completely clear, infuse an equal volume of 4%PFA solution. Dissect the tissues and organs and fix them overnight with 4%PFA at four degrees Celsius.To induce tissue decalcification. place the fixed heart tissue in 10 milliliters of EDTA solution on a shaking table at 37 degrees Celsius. Incubate for about two days and change the fluid daily.Place the fixed calvaria bones in 20 milliliters of 25%Quadrol solution for tissue decolorization. Incubate on a shaking table at 37 degrees Celsius for one day and change the fluid once. Next, place the tissue sequentially in 30%Tertiary-Butanol or TB solution.Then 50%TB solution, and then 70%TB solution to perform gradient degreasing. Subsequently dehydrate the samples and TB PEG solution for six hours on a shaking table at 37 degrees Celsius. Finally, place the completely dehydrated tissue in BB PEG solution on a shaking table at 37 degrees Celsius.After two to four hours of incubation, the tissue will turn transparent. The PEGASOS Tissue Clearing Method resulted in transparent tissue. Cleared tissue with whole mount EDU staining showed that the labeling was efficient and well preserved.In the control group, several EDU positive cells were diffusely distributed throughout the sutures. After one day of suture expansion, proliferating cells were observed in the middle and edges of the suture. As the suture widened, the number of proliferating cells decreased over time.The green labeled cells were small round cells in the bone marrow, which differed from the EDU positive suture cells.