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

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.