Half-segmental Diaphyseal Bone Defect Model in Rats for Evaluating Bone Substitute Performance in Load-bearing Regions

Shuhan Liu; Peng Yu; Jialu Jin; Yuchuan Luo; Yiyun Chen; Feifei Li; Yitian Wang; Ling Ye; Jiayi Wu

doi:10.3791/69211

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Method Article

Half-segmental Diaphyseal Bone Defect Model in Rats for Evaluating Bone Substitute Performance in Load-bearing Regions

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DOI:

10.3791/69211

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December 30th, 2025

Shuhan Liu¹^,² , Peng Yu¹ , Jialu Jin¹^,² , Yuchuan Luo¹^,² , Yiyun Chen¹^,² , Feifei Li¹ , Yitian Wang¹ , Ling Ye¹^,² , Jiayi Wu¹^,³

¹State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, ²Department of Cariology and Endodontics, West China Hospital of Stomatology, Sichuan University, ³Department of Jinjiang Outpatient, West China Hospital of Stomatology, Sichuan University

Summary

This study established a rat femoral half-segmental defect model to evaluate the mechanical and osteogenic performance of bone substitute materials under load-bearing conditions without the use of internal or external fixation.

Abstract

Accurate preclinical evaluation of bone substitute materials demands models that not only support tissue regeneration but also reflect the mechanical challenges of physiological load-bearing conditions. However, most conventional animal bone defect models fall short in this regard. For example, calvarial defect models, though widely used, are created in non-load-bearing regions. Similarly, drill-hole or partial defects in long bones primarily focus on localized healing, lacking sufficient load transfer to assess mechanical support. This study established a standardized half-segmental diaphyseal bone defect model in the rat femur to assess both the mechanical and biological performance of bone substitute materials in a load-bearing context without the use of fixation devices. Using a high-speed handpiece and a specialized cylindrical dental bur, a semi-cylindrical defect measuring 4 mm in length and 1.5 mm in radius was created in the lateral midshaft of the femur. Two representative materials with distinct properties -- Gelatin methacryloyl (GelMA) hydrogel and 3D-printed polymethyl methacrylate (3DP-PMMA) -- were implanted to validate the model's capacity to differentiate the mechanical properties of materials. Radiographic, histological, and immunofluorescent analyses performed at 4 weeks post-implantation revealed that, due to inadequate mechanical support, GelMA implantation led to malunion with fibrous tissue predominance and limited bone integration. In contrast, 3DP-PMMA facilitated the organization of new bone formation, periosteal continuity, and the recruitment of Piezo type mechanosensitive ion channel component 1-expressing (Piezo1⁺) and leptin receptor-expressing (LepR⁺) cells. This model provides a practical and reproducible platform for assessing the load-bearing capacity, osteoinductivity, and osseointegration potential of biomaterials. It can serve as a valuable tool for screening candidate materials for orthopedic applications, such as long bone reconstruction or fracture repair.

Introduction

Under physiological conditions, bone plays a central role in maintaining the structural integrity and mechanical function of the body. It supports body weight, enables movement, and protects internal organs. When bone is damaged due to trauma, tumor resection, or infection, especially in load-bearing regions, the mechanical continuity is interrupted. This disruption often leads to complications such as bone deformity, reduced mobility, limb instability, chronic pain, and even life-threatening issues from severe fractures and hemorrhage¹^,². Nowadays, bone substitutes such as bone grafting and biomaterials have ....