Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

Summary

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Abstract

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample's defect area was evaluated using micro-CT, hematoxylin and eosin (H&E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model's healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

Introduction

Fractured and defective bone often results from trauma, tumors, inflammation, and congenital malformations^1,2. Although bone tissue in young healthy individuals typically possesses robust regenerative abilities³, defects exceeding a critical size or healing impediments due to systemic diseases (e.g., diabetes, osteoporosis, and infections) may still lead to complications such as bone discontinuity or impaired healing⁴. To address this clinical challenge, bone grafting or biomaterials are commonly used to replace severely defective bone or to reconstruct large bon....

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). Sprague-Dawley rats (male, body weight 300 g) were used for the present study.

1. Presurgical preparation

1. Instrument preparation
   1. Refer to Figure 1A for the surgical instruments used in this study: electric shaver, tissue scissors, ophthalmic scissors, ophthalmic forceps, di.......

Discussion

Preclinical animal models are vital for examining bone healing and the influence of biomaterials on bone regeneration. This protocol illustrates a femoral box-cavity defect model replicating the intramembranous bone formation process associated with clinical bone regeneration. The defect area was intraoperatively standardized using a pre-marked oral probe. Micro-CT and histopathological staining results showed progressive healing over 6 weeks, with thickened periosteum and new trabecular bone formation, followed by dense.......

Materials

Name	Company	Catalog Number	Comments
1.2 mm slow speed ball drill	Dreybird Medical Equipment Co., Ltd.	RA3-012	For preparation of box cavity defects
3.0 suture	Chengdu Shifeng Co., Ltd.	None	For suturing wounds
4% paraformaldehyde	Biosharp	BL539A	For fix the femoral specimens
Cotton balls	Haishi Hainuo Group Co.,  Ltd.	20120047	For skin sterilization and cleaning of surgical field
Cotton sticks	Lakong Medical Devices Co., Ltd.	M6500R	For skin disinfection
Dental technician grinding machine	Marathon	N3-140232	For preparation of box cavity defects
Disposable scalpel	Hangzhou Huawei Medical Supplies Co., Ltd.	20100227	For creating skin incisions as well as to sharply separate muscle tissue
Electric shaver	JASE	BM320210	Removal of hair tissue from the surgical area
Hematoxylin and Eosin Stain kit	Biosharp	C1005	For the histological analysis of the specimens
Masson’s Trichrome Stain Kit	Solarbio	G1340	For the histological analysis of the specimens
Micro CT	Scanco medical ag	µCT 45	For analyzing the healing of defects in femoral samples
Needle holder	Chengdu Shifeng Co., Ltd.	None	For suture-holding needles
Olympus Research Grade Whole Slide Scanning System VS200	Chengdu Knowledge Technology Co.	VS200	For analyzing the results of HE staining and Masson staining
Ophthalmic forceps	Chengdu Shifeng Co., Ltd.	None	For clamping skin, muscle tissue
Ophthalmic scissors	Chengdu Shifeng Co., Ltd.	None	For forming a skin incision approach
Oral low-speed handpiece	Marathon	Y221101003	For preparation of box cavity defects
Oral probe	Shanghai Sangda Medical Insurance Co., Ltd.	20000143	For measuring the diameter of defects
Periosteal separator	Chengdu Shifeng Co., Ltd.	None	For blunt separation of muscle tissue
Sprague–Dawley rats	Byrness Weil Biotech Ltd	None	For the establishment of femoral bone boxy cavitary defect
Tissue scissors	Chengdu Shifeng Co., Ltd.	None	For forming a skin incision approach