This study introduced a rat model for preclinical investigation of magnesium-based biodegradable materials in bone fracture conditions, allowing longitudinal in vivo monitoring of fracture healing and implant degradation with multimodal imaging.

Histochemical staining of paraffin-embedded decalcified bone samples is commonly used in preclinical research of musculoskeletal diseases, enabling the visualization of multiple tissue components by the application of chromogens. The purpose of this study was to introduce a novel multicolor staining protocol involving optimized chemical reagents and procedure, allowing the identification of high-mineralized bone, low-mineralized fracture callus, cartilage and skeletal muscle fibers simultaneously. Fractured femur and healthy tail vertebra samples from adult male Sprague–Dawley rats were decalcified with EDTA and formic acid, respectively, followed by paraffin embedding, tissue sectioning and multicolor staining. Conventional Movat’s pentachrome and safranin O / fast green staining were conducted in parallel for comparison. Immunohistochemical staining of collagen type-X and micro-CT analysis were included to further validate the efficacy of the staining method. The multicolor staining allowed visualization of major musculoskeletal tissue components in both types of decalcified samples, providing quality outcomes with fewer chemical reagents and simplified procedures. Immunohistochemical staining demonstrated its capacity for identification of the endochondral ossification process during fracture healing. Micro-CT imaging validated the staining outcome for high-mineralized skeletal tissue. The application of the multicolor staining may facilitate future preclinical research involving decalcified paraffin-embedded samples.

Though surgical techniques profoundly influence in vivo experiments, significant heterogeneity exists in current surgeries for inducing rat femoral bone defects. Such variations reduce the reproducibility and comparability of preclinical studies, and are detrimental to clinical translation. The purposes of this study were: (1) to conduct a systematic review of rat femoral defect models, summarizing and analyzing the surgical techniques; (2) to analyze surgical design and potential pitfalls via 3D anatomy and virtual surgeries for fostering future precision research; and (3) to establish a surgical classification system, for improving the reproducibility and comparability among studies, avoiding unnecessary repetitive experiments. The online database PubMed was searched to identify studies from January 2000 to June 2022 using keywords, including rat, femur, bone defect. Eligible publications were included for a review of surgical methods. Anatomical analysis and virtual surgeries were conducted based on micro-CT reconstruction of the rat femur for further investigation and establishment of a classification system. A total of 545 publications were included, revealing marked heterogeneity in surgical methods. Four major surgical designs were reported for inducing defects from the proximal to distal femur: bone tunnel, cortical window, segmental defect, and wedge-shaped defect. Anatomical analysis revealed potential pitfalls hindering efficient clinical translation. A classification system was established according to the anatomical region, surgical design, and fixation devices. This systematic review in combination with 3D analysis and virtual surgery provides a general overview of current surgical approaches to inducing femoral defects in rats, and establishes a surgical classification facilitating preclinical research of quality and translational value.