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

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.......

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.......

Materials

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