rat femoral diaphysis box-cavity creation

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

Fractured and defective bone often results from trauma, tumors, inflammation, and congenital malformations1,2. 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 bone segments. However, these treatments have limitations. For instance, although considered the gold standard, autologous bone grafting suffers from restricted donor supply and potential donor site complications5,6. Allografts also present certain risks, such as immune-mediated rejection, potential transmission of diseases, and negative impacts on the biomechanical and biological properties of the graft7.
Recent years have witnessed a surge in research focusing on bone defect healing mechanisms. The use of alternative biomaterials and advancements in tissue engineering have emerged as prominent topics within the domain of bone regeneration8. Before these biomaterials can be applied to human therapy, they must be tested in vitro and in vivo to ensure their efficacy and safety. However, the reduced complexity of in vitro environments and the absence of immune and inflammatory responses limit the evaluation of various biomaterials in vitro. Consequently, the establishment of animal models for various types of bone tissue defects is needed9. Animal models allow the evaluation of biomaterials under different loading conditions, facilitate understanding of species-specific bone characteristics, and provide insight into the similarity between animal models and human clinical situations. These advantages are essential for studying bone-scaffold interactions and translating research findings into clinical practice9,10.
Currently, mechanical bone defect animal models are widely used to validate the performance of biomaterials, with cranial bone defect models and segmental bone defect models being the most commonly applied methods11. Segmental bone defect models, often utilized to mimic severe long bone or tibial trauma ending in bone nonunion, are advantageous due to their uniform dimensions and defined anatomical positions, simplifying radiological or histological evaluations of new bone formation and revascularization. However, these models require metal implants to stabilize bilateral fracture segments and necessitate a complex healing process involving both endochondral and intramembranous ossification12. On the other hand, calvarial bone defect models have become a primary screening tool for evaluating biomaterials due to their standardized defect diameters, convenient surgical access, and the supportive function of dura mater and soft tissue13. Although they are widely used for modeling intramembranous bone formation in clinically relevant scenarios, they are unsuitable for evaluating bone healing under biomechanical loading conditions due to their non-load-bearing nature during the healing process14.
To address these limitations, we established a box-cavity cortical bone defect model in the femoral diaphysis tissue of rats. Utilizing micro-computed tomography (CT) three-dimensional (3D) reconstruction, and histopathological staining (Hematoxylin and eosin [HE] and Masson), we analyzed the healing process of this model under hemostasis conditions. We aim to offer fresh insights for evaluating biomaterial performance under biomechanical loading conditions and for studying the bioengineering and mechanism of bone regeneration vis-&#224;-vis intramembranous ossification.

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

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

Various animal models with bone defects are applied in exploring the therapeutic potential of tissue engineering for bone healing. However, most of them are unable to generate standard defects under biomechanical loading conditions, which is important in evaluating the bone healing effect of bio materials. Compared to traditional bone defect models, the recent box cavity cortical bone defect model offers significant advancements.It not only provides a standardized bone defect size for more accurate quantification of bone healing, but also allows for the assessment of bio materials under biomechanical loading conditions without the need for additional fixation. Our protocol provides a comprehensive and generalizable approach for investigating intramembranous bone formation within in vivo study of bone regeneration models. This is particularly beneficial for the field of bone tissue engineering.

Rat Femoral Diaphysis Box-Cavity Creation

To begin place the anesthetized rat in a lateral recumbent position on the sterile surgical table. Remove the lower limb hairs with an electric shaver. Disinfect the surgical area in a circular motion with 5%iota four solution, followed by 75%alcohol.After locating the proximal and distal femur, make a 2.5 centimeter incision along the long axis of the femur to cut through the skin tissue. Using ophthalmic forceps and tissue scissors, separate the skin layer from the fascia. Expose the lateral approach to the femur through the biceps femurs and lateral femoral muscles.Locate the intersection of the two muscle septa and carefully separate with a disposable surgical blade along the muscle border until the femoral surface is reached. Next, apply a periosteal separator to bluntly separate the femoral surface muscles and expose the middle of the femoral diaphysis. Using a sterile marker pen, mark the area of the defect site on the mids surface of the femoral diaphysis.Next, using an oral low speed handpiece drill a small hole perpendicular to the bone surface at the marked site, destroying the periosteum and bone cortex to depth reaching the bone marrow cavity. Expand the hole in all directions to achieve a box cavity shape. Now hold a labeled oral probe parallel to the edge of the defect to determine the defect diameter and morphology.Suture to close the muscle and skin layers. Finally, disinfect the surgical area with 5%iota four from the inside out.

rat-femoral-diaphysis-box-cavity-creation

Research

JoVE Journal

Medicine

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&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;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&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

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.

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...