Assessment of Bone Regeneration in Rabbit Calvarial Defects Treated With Mineralized Collagen-Based Scaffolds Using...

Cranial bone regeneration requires coordinated interactions among host cells, extracellular matrices, and the local biomechanical environment to restore both mineralized tissue and structural protection of the brain. Mineralized collagen-glycosaminoglycan (MC-GAG) scaffolds recapitulate key features of native bone matrix and have demonstrated osteogenic potential in preclinical calvarial defect models; yet, robust, quantitative methods are needed to evaluate defect bridging, mineral density, and microarchitectural maturation in vivo. In vivo microcomputed tomography (microCT) enables nondestructive, three-dimensional (3D) assessment of craniofacial healing with spatial detail sufficient to characterize mineral distribution and structural connectivity across large defects. Here, we describe an integrated workflow that combines scaffold implantation in a rabbit critical-sized calvarial defect model with standardized in vivo microCT imaging and 3D computational rendering. The framework includes scaffold fabrication and preparation, surgical creation of a critical-sized calvarial defect with scaffold placement, and in vivo microCT imaging at a single endpoint (6 months post-surgery). Image datasets are visualized and analyzed using reproducible segmentation software (i.e., ORS Dragonfly) to examine defect repair and extract quantitative intensity- and volume-based metrics, including mineralized tissue volume, mineral density, and microarchitectural parameters. This standardized approach supports objective, image-based comparison of scaffold designs and treatment strategies, facilitating rigorous evaluation of cranial bone regeneration and accelerating the development of bone-regenerative biomaterials. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fabrication of mineralized collagen-based scaffolds Basic Protocol 2: Surgical creation of rabbit calvarial defects with scaffold implantation and in vivo microCT scanning Basic Protocol 3: In vivo microCT data visualization, rendering, and analysis of MC-GAG scaffolds-implanted rabbit calvarial bone defect.