Longitudinal in vivo micro-computed tomography (micro-CT) is of interest to non-invasively capture the healing process of individual animals in preclinical fracture healing studies. However, it is not known whether longitudinal imaging itself has an impact on callus formation and remodeling. In this study, a scan group received weekly micro-CT measurements (week 0-6), whereas controls were only scanned post-operatively and at week 5 and 6. Registration of consecutive scans using a branching scheme (bridged vs. unbridged defect) combined with a two-threshold approach enabled assessment of localized bone turnover and mineralization kinetics relevant for monitoring callus remodeling. Weekly micro-CT application did not significantly change any of the assessed callus parameters in the defect and periosteal volumes. This was supported by histomorphometry showing only small amounts of cartilage residuals in both groups, indicating progression towards the end of the healing period. Also, immunohistochemical staining of Sclerostin, previously associated with mediating adverse radiation effects on bone, did not reveal differences between groups. The established longitudinal in vivo micro-CT-based approach allows monitoring of healing phases in mouse femur defect models without significant effects of anesthesia, handling and radiation on callus properties. Therefore, this study supports application of longitudinal in vivo micro-CT for healing-phase-specific monitoring of fracture repair in mice.

Thorough preclinical evaluation of functionalized biomaterials for treatment of large bone defects is essential prior to clinical application. Using in vivo micro-computed tomography (micro-CT) and mouse femoral defect models with different defect sizes, we were able to detect spatio-temporal healing patterns indicative of physiological and impaired healing in three defect sub-volumes and the adjacent cortex. The time-lapsed in vivo micro-CT-based approach was then applied to evaluate the bone regeneration potential of functionalized biomaterials using collagen and bone morphogenetic protein (BMP-2). Both collagen and BMP-2 treatment led to distinct changes in bone turnover in the different healing phases. Despite increased periosteal bone formation, 87.5% of the defects treated with collagen scaffolds resulted in non-unions. Additional BMP-2 application significantly accelerated the healing process and increased the union rate to 100%. This study further shows potential of time-lapsed in vivo micro-CT for capturing spatio-temporal deviations preceding non-union formation and how this can be prevented by application of functionalized biomaterials. This study therefore supports the application of longitudinal in vivo micro-CT for discrimination of normal and disturbed healing patterns and for the spatio-temporal characterization of the bone regeneration capacity of functionalized biomaterials.