Osteoporosis is the most common bone disease. However, the mechanism of osteoporosis-induced alterations in bone is still unclear. The aim of this study was to investigate the effects of osteoporosis on the structural, densitometric and mechanical properties of the whole tibia using in vivo μCT imaging, spatiotemporal analysis and finite element modeling. Twelve C57Bl/6 female mice were adopted. At 14 weeks of age, half of the mice were ovariectomized (OVX), and the other half were SHAM-operated. The whole right tibia was scanned using an in vivo μCT imaging system at 14, 16, 17, 18, 19, 20, 21 and 22 weeks. The image datasets were registered in order to precisely quantify the bone properties. The results showed that OVX led to a significant increase in the endosteal area across the whole tibia 4 weeks after OVX intervention but did not have a significant influence on the periosteal area. Additionally, the bone volume and mineral content significantly decreased only in the proximal regions, but these decreases did not have a significant influence on the stiffness and failure load of the tibia. This study demonstrated the application of a novel spatiotemporal approach in the comprehensive analysis of bone adaptations in the spatiotemporal space.

In vivo µCT imaging allows for high-resolution, longitudinal evaluation of bone properties. Based on this technology, several recent studies have developed in vivo dynamic bone histomorphometry techniques that utilize registered µCT images to identify regions of bone formation and resorption, allowing for longitudinal assessment of bone remodeling. However, this analysis requires a direct voxel-by-voxel subtraction between image pairs, necessitating rotation of the images into the same coordinate system, which introduces interpolation errors. We developed a novel image transformation scheme, matched-angle transformation (MAT), whereby the interpolation errors are minimized by equally rotating both the follow-up and baseline images instead of the standard of rotating one image while the other remains fixed. This new method greatly reduced interpolation biases caused by the standard transformation. Additionally, our study evaluated the reproducibility and precision of bone remodeling measurements made via in vivo dynamic bone histomorphometry. Although bone remodeling measurements showed moderate baseline noise, precision was adequate to measure physiologically relevant changes in bone remodeling, and measurements had relatively good reproducibility, with intra-class correlation coefficients of 0.75-0.95. This indicates that, when used in conjunction with MAT, in vivo dynamic histomorphometry provides a reliable assessment of bone remodeling.

In this study we established an image analysis scheme for the investigation of cortical and trabecular bone development during skeletal growth and tested this concept on in vivo μCT images of rats. To evaluate its efficacy, we applied the technique to young (1-month-old) and adult (3-month-old) rat tibiae with vehicle (Veh) or intermittent parathyroid hormone (PTH) treatment. By overlaying 2 sequential scans based on their distinct trabecular microarchitecture, we calculated the linear growth rate of young rats to be 0.31 mm/day at the proximal tibia. Due to rapid growth (3.7 mm in 12 days), the scanned bone region at day 12 had no overlap with the bone tissue scanned at day 0. Instead, the imaged bone region at day 12 represented newly generated bone tissue from the growth plate. The new bone of the PTH-treated rats had significantly greater trabecular bone volume fraction, number, and thickness than those of the Veh-treated rats, indicating PTH\'s anabolic effect on bone modeling. In contrast, the effect of PTH on adult rat trabecular bone was found to be caused by PTH\'s anabolic effect on bone remodeling. The cortical bone at the proximal tibia of young rats also thickened more in the PTH group (23%) than the Veh group (14%). This was primarily driven by endosteal bone formation and coalescence of trabecular bone into the cortex. This process can be visualized by aligning the local bone structural changes using image registration. As a result, the cortex after PTH treatment was 31% less porous, and had a 22% greater polar moment of inertia compared to the Veh group. Lastly, we monitored the longitudinal bone growth in adult rats by measuring the distance of bone flow away from the proximal tibial growth plate from 3 months to 19 months of age and discovered a total of 3.5mm growth in 16 months. It was demonstrated that this image analysis scheme can efficiently evaluate bone growth, bone modeling, and bone remodeling, and is ready to be translated into a clinical imaging platform.

Current osteoporosis treatments improve bone mass by increasing net bone formation: anti-resorptive drugs such as bisphosphonates block osteoclast activity, while anabolic agents such as parathyroid hormone (PTH) increase bone remodeling, with a greater effect on formation. Although these drugs are widely used, their role in modulating formation and resorption is not fully understood, due in part to technical limitations in the ability to longitudinally assess bone remodeling. Importantly, it is not known whether or not PTH-induced bone formation is independent of resorption, resulting in controversy over the effectiveness of combination therapies that use both PTH and an anti-resorptive. In this study, we developed a μCT-based, in vivo dynamic bone histomorphometry technique for rat tibiae, and applied this method to longitudinally track changes in bone resorption and formation as a result of treatment with alendronate (ALN), PTH, or combination therapy of both PTH and ALN (PTH+ALN). Correlations between our μCT-based measures of bone formation and measures of bone formation based on calcein-labeled histology (r=0.72-0.83) confirm the accuracy of this method. Bone remodeling parameters measured through μCT-based in vivo dynamic bone histomorphometry indicate an increased rate of bone formation in rats treated with PTH and PTH+ALN, together with a decrease in bone resorption measures in rats treated with ALN and PTH+ALN. These results were further supported by traditional histology-based measurements, suggesting that PTH was able to induce bone formation while bone resorption was suppressed.

Daily injections of parathyroid hormone (PTH) are the only FDA-approved anabolic treatment for osteoporosis; however PTH is only clinically approved for treatment periods of up to 24months. To enhance its anabolic effect, combining PTH with anti-resorptive therapy was proposed and expected to maximize the effectiveness of PTH. The current study aimed to elucidate structural mechanisms through which combination therapy can further improve bone strength over a limited treatment window of 12days, to more closely examine the early phase of the anabolic window. We examined 30 female rats treated with either vehicle (Veh), alendronate (ALN), PTH, or both PTH and ALN (PTH+ALN). Standard and individual trabecula segmentation (ITS)-based microstructural analyses were performed using in vivo micro-computed tomography. We found an increase in BV/TV in all treatments with the highest in the PTH+ALN group. Tb.Th* increased in both PTH and PTH+ALN groups well beyond that of the Veh or ALN group. SMI decreased in all treatments with PTH+ALN having the greatest tendency toward plate-like structures. ITS confirmed the trend toward more plate-like structures with increased plate Tb.N* and increased plate-to-rod ratio that was most pronounced in the PTH+ALN group. Using image-based finite element analysis, we demonstrated that stiffness increased in all treatment groups, again with the largest increase in the PTH+ALN group, indicating the resulting structural implications of increased plate-like structure. Static and dynamic bone histomorphometry and a serum resorption marker confirmed that PTH+ALN significantly increased bone formation activities and suppressed bone resorption activities. Overall the results indicate that PTH+ALN treatment has an additive effect due to a preferential increase in plate-like structures.

In the recent decade, in vivo μCT scanners have become available to monitor temporal changes in rodent bone in response to diseases and treatments. We investigated short-term and long-term precision of in vivo μCT measurements of trabecular bone density, microstructure and stiffness of rat tibiae and tested whether they can be improved by 3D image registration. Rats in the short-term precision group underwent baseline and follow-up scans within the same day (n = 15) and those in the long-term precision group were scanned at day 0 and day 14 (n = 16) at 10.5 μm voxel size. A 3D image-registration scheme was applied to register the trabecular bone compartments of baseline and follow-up scans. Prior to image registration, short-term precision ranged between 0.85% and 2.65% in bone volume fraction (BV/TV), trabecular number, thickness, and spacing (Tb.N*, Tb.Th*, Tb.Sp*), trabecular bone mineral density and tissue mineral density (Tb.BMD, and Tb.TMD), and was particularly high in structure model index (SMI), connectivity density (Conn.D), and stiffness (4.29%-8.83%). Image registration tended to improve the short-term precision, but the only statistically significant improvement was in Tb.N*, Tb.TMD, and stiffness. On the other hand, unregistered comparisons between day-0 and day-14 scans suggested significant increases in BV/TV, Tb.N*, Tb.Th*, Conn.D, and Tb.BMD and decrease in Tb.Sp* and SMI. However, the percent change in each parameter from registered comparisons was significantly different from unregistered comparisons. Registered results suggested a significant increase in BV/TV, Tb.BMD, and stiffness over 14 days, primarily caused by increased Tb.Th* and Tb.TMD. Due to the continuous growth of rodents, the direct comparisons between the unregistered baseline and follow-up scans were driven by changes due to global bone modeling instead of local remodeling. Our results suggested that 3D image registration is critical for detecting changes due to bone remodeling activities in rodent trabecular bone by in vivo μCT imaging.