The Establishment of Calvarial Suture-Bony Composite Defects in Rats: A Standardized Model for Suture-Regenerative Therapy Investigation

Summary

We describe the detailed surgical procedures of calvarial suture-bony composite defects in rats, alongside the investigations into the short-term and long-term prognoses of the model. We aim to construct a standardized model for developing suture-regenerative therapies.

Abstract

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development (the latter occurs in the developing cranium). However, the lack of a standardized model hinders progress in investigating suture-regenerative therapies and poses challenges for conducting comparative analyses across distinct studies. To address this issue, the current protocol describes the detailed modeling process of calvarial suture-bony composite defects in rats.

The model was generated by drilling full-thickness rectangular holes measuring 4.5 mm × 2 mm across the coronal sutures. The rats were euthanized, and the cranium samples were harvested postoperatively at day 0, week 2, week 6, and week 12. µCT results from samples collected immediately post-surgery confirmed the successful establishment of the suture-bony composite defect, involving the removal of the coronal suture and the adjacent bone tissues.

Data from the 6^th and 12^th postoperative weeks demonstrated a natural healing tendency for the defect to close. Histological staining further validated this trend by showing increased mineralized fibers and new bone at the defect center. These findings indicate progressive suture fusion over time following calvarial defects, underscoring the significance of therapeutic interventions for suture regeneration. We anticipate that this protocol will facilitate the development of suture-regenerative therapies, offering fresh insights into the functional restoration of calvarial defects and reducing adverse outcomes associated with suture loss.

Introduction

Cranial sutures are dense fibrous connections between cranial bones, acting as joints to facilitate slight skull movement and providing a protective cushion for the brain under pressure¹. In recent years, increased research has highlighted the pivotal role of cranial sutures in skull development, craniofacial homeostasis, and inherent osteo-reparative potential^{2,3,4,5,6,7,8}. During periods of growth and development....

Protocol

All animal procedures in this study were reviewed and approved by the Ethical Committee of the West China School of Stomatology, Sichuan University (WCHSIRB-D-2021-597). A total of 12 (3 rats at each of the four time points) Sprague-Dawley (SD) rats (male, 300 g, 8 weeks old) were obtained from a commercial source (see Table of Materials).

1. Presurgical preparation

1. Surgical items
   1. Prepare surgical instruments displayed in

Discussion

Conventional calvarial defect models, whether involving cranial sutures or not, primarily concentrate on the repair of hard tissue, often neglecting the vital regeneration of suture mesenchyme^19,20. In suture regeneration research, prior models, like those by Mardas et al.^15,16, utilizing a trephine bur to create a 5 mm circular defect across the sagittal suture of rats, resulted in substantial hard tissu.......

Materials

Name	Company	Catalog Number	Comments
4% paraformaldehyde	Biosharp	BL539A
2% Iodophor solution	Chengdu Jinshan Chemical Reagent Co., Ltd.	None
75% Ethanol	Chengdu Jinshan Chemical Reagent Co., Ltd.	None
Cotton balls	Haishi Hainuo Group Co., Ltd.	None
Cotton swabs	Lakong Medical Devices Co.,	None
Curved forceps	Chengdu Shifeng Co., Ltd.	None
Dataviewer and Ctan software for residual defect volume assessments	Bruker	None
Dental low-speed round burs	Dreybird Medical Equipment Co., Ltd.	RA3-012 RA1-008
Disposable sterile scalpel	Hangzhou Huawei Medical Supplies Co., Ltd.	None
Disposable syringes (22 G)	Chengdu Shifeng Co., Ltd.	SB1-089(IX)
Electric shaver	JASE	BM320210
Ethylene Diamine Tetraacetic Acid (EDTA)	BioFroxx	1340GR500
Hematoxylin and Eosin Stain Kit	Biosharp	BL700B
Irrigation needle (23 G)	Sichuan New Century Medical Polymer Products Co., Ltd.	None
Low-speed handpiece	Guangzhou Dental Guard Technology Co., Ltd.	None
Masson’s Trichrome Stain Kit	Solarbio	G1340
Medical non-woven fabrics	Henan Yadu Industrial Co., Ltd.	None
Micro-computed tomography (µCT)	Scanco Medical AG	µCT45
Mimics 20.0 for cross-sectional images	Materialise	None
Needle holders	Chengdu Shifeng Co., Ltd.	None
Periosteal elevator	Chengdu Shifeng Co., Ltd.	None
Saline solution	Sichuan Kelun Pharmaceutical Co., Ltd.	None
Scanco medical visualizer software for 3D image reconstruction	Scanco Medical AG	None
SPSS Statistics 20.0 for statistical analysis	IBM	None
Sprague-Dawley rats	Byrness Weil Biotech Ltd	None
Straight Scissors	Chengdu Shifeng Co., Ltd.	None
Surgical Motor	MARATHON	N3-140232
Surgical sutures (3-0 monofilament)	Hangzhou Huawei Medical Supplies Co., Ltd.	None