Comprehensive guideline for X-ray microtomography-guided bone analysis and histology applied to the assessment of defect repair in long and flat bones

Bone regeneration studies require reliable analytical tools capable of assessing both microstructural and cellular aspects of tissue repair. This article presents a comprehensive methodological guide that integrates microcomputed tomography (micro-CT) and histological analyses for standardized evaluation of bone defect repair in rodent models, focusing on the tibia (long bone) and calvaria (flat bone). We describe detailed surgical protocols for defect creation, imaging workflows for micro-CT acquisition, reconstruction, and morphometric quantification, alongside histological processing and semi-quantitative scoring of tissue parameters. Illustrative results demonstrate how micro-CT enables three-dimensional, non-destructive quantification of mineralized tissue, while histology provides complementary insights into non-mineralized structures, vascularization, and cellular organization. The combined use of these techniques enhances reproducibility, ensures methodological consistency, and allows for robust comparisons across studies. By establishing a standardized workflow, this protocol supports translational research in bone repair and offers a valuable resource for evaluating regenerative strategies and biomaterials.