Spatio-temporal characterization of fracture healing patterns and assessment of biomaterials by time-lapsed in vivo micro-computed tomography

Abstract

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.

Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging

“…S1 ). Similar to observations from a previous femur defect study 25 , nest building started on the day of surgery and all animals were found in groups in the nest in the morning of post-operative day 1. In both groups, social interaction between mice and nesting behaviour during the pre-loading healing period (week 0–3) and the loading period (week 3–7) did not differ from pre-surgical observations.…”

“…Compared to histology-based determination of the bone formation rate via in vivo application of fluorescent dyes (e.g. calcein, alizarin), our micro-CT-based monitoring approach with registration of consecutive scans also allows for assessment of the bone resorption rate in individual animals, which was shown to be particularly suited for the characterization of the remodelling phase during the fracture healing process 25 , 26 . The most striking loading-mediated effect in the current study was the prompt and significant reduction in bone resorption rate (week 3–4 vs. week 2–3), which was seen in the central and periosteal VOIs (DC, FP) as early as 1 week after loading (Fig.…”

“…We confirm that all methods were carried out in accordance with relevant guidelines and regulations (Swiss Animal Welfare Act and Ordinance (TSchG, TSchV)) and reported considering ARRIVE guidelines. Animal experiments were performed using previously established protocols for osteotomy surgery 25 , in vivo micro-CT imaging 26 , analgesia/anaesthesia 25 , 26 and post-operative monitoring 25 . To study adult fracture healing female 12 week-old C57BL/6J mice were purchased from Janvier (Saint Berthevin Cedex, France) and housed in the animal facility of the ETH Phenomics Center (EPIC; 12 h:12 h light–dark cycle, maintenance feed (3437, KLIBA NAFAG, Kaiseraugst, Switzerland), 5 animals/cage) for 8 weeks.…”

“…By applying time-lapsed in vivo micro-CT and animal-specific real-time micro-finite element (RTFE) analysis, the developed femur defect loading model allows scaling of loading settings based on strain distribution in the mineralized callus 24 , thereby considering individual differences in healing speed. To follow the healing progression in each animal, a recently developed longitudinal in vivo micro-CT based approach was applied, which was previously shown to capture the different healing phases and to discriminate physiological and impaired healing patterns 25 , without significant radiation-associated effects on callus properties 26 . By registering consecutive scans of the defect region for each animal and implementing a two-threshold approach, data on bone turnover and mineralization kinetics can be obtained, which is particularly important for targeting callus remodelling under different conditions.…”

Individualized cyclic mechanical loading improves callus properties during the remodelling phase of fracture healing in mice as assessed from time-lapsed in vivo imaging

“…By applying time-lapsed in vivo micro-CT and animal-specific real-time micro-finite element (RTFE) analysis, the developed femur defect loading model allows scaling of loading settings based on strain distribution in the callus 16 , thereby considering individual differences in healing speed. To follow the healing progression in each animal, a recently developed longitudinal in vivo micro-CT based approach was applied, which was previously shown to capture the different healing phases and to discriminate physiological and impaired healing patterns 17 , without significant radiation-associated effects on callus properties 18 . By registering consecutive scans of the defect region for each animal and implementing a two-threshold approach, data on bone turnover and mineralization kinetics can be obtained, which is particularly important for targeting callus remodelling under different conditions.…”