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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 "river bank", 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....

Protocol

All experimental procedures described here were approved by the Animal Care Committee of Shanghai Ninth People'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

  1. Preparation of two retention holders.
    1. Use 0.014'' Australian wire or stainless steel wir.......

Representative Results

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

Discussion

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.......

Acknowledgements

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's Hospital, Shanghai Jiao Tong University School of Medicine;Fundamental research program funding of Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine (JYZZ154).

....

Materials

NameCompanyCatalog NumberComments
37% Acid etchingXihubiomE10-02/1807011
Alizarin redSigma-AldrichA3882
AUSTRALIAN WIREA.J.WILCOCK0.014''
Benzyl benzoateSigma-AldrichB6630
Calcein greenSigma-AldrichC0875
Copper(II) sulfate, anhydrousSangon BiotechA603008
DynamometerSanliangSF-10N
EDTASigma-AldrichE9884
EdUInvitrogenE104152
Laser Confocal MicroscopeLeicaSP8
PBSSangon BiotechE607008
PEG-MMA 500Sigma-Aldrich447943
PFASigma-AldrichP6148 
pH MetersMettler ToledoS220
QuadrolSigma-Aldrich122262
Sodium AscorbateSigma-AldrichA4034
Sodium bicarbonateSangon BiotechA500873
Sodium chlorideSangon BiotechA610476
Sodium hydroxideSigma-AldrichS5881
SpringTAOBAO0.2*1.5*1*7
Sulfo-Cyanine3 azideLumiprobeA1330
tert-ButanolSigma-Aldrich360538 Protect from light. Do not freeze.
Transbond MIP
Moisture Insensitive Primer
3M Unitek712-025
Transbond XT
Light Cure Adhesive Paste
3M Unitek712-035
TriethanolamineSigma-AldrichV900257
Tris-buffered salineSangon BiotechA500027

References

  1. Opperman, L. A. Cranial sutures as intramembranous bone growth sites. Developmental Dynamics. 219 (4), 472-485 (2000).
  2. Thenier-Villa, J. L., Sanromán-Álvarez, P., Miranda-Lloret, P., Plaza Ramírez, M. E.

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3D VisualizationBone RemodelingSuture ExpansionMouse ModelPEGASOS Tissue ClearingStem CellsOrthopedic TreatmentCraniofacial SuturesIntramembranous OssificationBone RemodelingEdU StainingCalcium Chelating

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