To study the short- and long-term effects of estrogen deficiency on trabecular bone, three-dimensional measurements of proximal tibiae of ovariectomized rats were performed by micro-computed tomography (MicroCT). New three-dimensional (3D) techniques were employed to characterize the trabecular architecture from 0 to 110 days post-ovariectomy (OVX). These new methods no longer assume a plate or rod model of bone, but calculate trabecular thickness, separation, and number and their distribution by placing maximal spheres into the 3D representation of the structure. The model type of bone was quantified with the Structure Model Index (SMI). Utilizing these methods we found a rapid loss of trabecular bone in the first week after OVX. After the first week bone mass declined further, although the rate of loss was lower. In addition there was a complete change in model type from plate-like to rod-like within 7 days post-OVX, and then a very constant SMI after 12 days. After an initial thinning of trabecular structure, further bone loss seems to occur through removal of trabeculae, while the trabecular plate thickness remains constant. The heterogeneity of the network could be quantified by intra-individual standard deviation of local separations, which showed a stair-like progression, with a plateau between 12 and 60 days post-OVX. This study provides new insights into ovariectomy-related changes in cancellous bone structure evaluated by 3D MicroCT. In addition, these data suggest that the rapid change of model from plate-like to rod-like post-OVX may potentially introduce biases in the parameters that are determined using model-based algorithms, and these biases may modify the impact of age-related or therapeutic changes.

This study evaluated the effect of Cenestin, a synthetic conjugated estrogens product, on the maintenance of trabecular bone microarchitecture, bone strength, and of bone turnover in the ovariectomized (ovx) rat model. Two doses of Cenestin were chosen in an attempt to approximate the equivalent human oral doses of 0.3 mg and 0.625 mg. Forty-nine 6-month-old retired female breeder Sprague-Dawley rats were randomly assigned to one of four groups: (1) sham-operated + vehicle; (2) ovx + vehicle; (3) ovx + day 1 post-ovariectomy Cenestin (8.12 mg/kg); (4) ovx + day 1 post-ovariectomy Cenestin (16.24 mg/kg) for 8 weeks. Trabecular structure of the right proximal tibia of each rat was imaged noninvasively by microCT. A compression test to induce a tibial plateau fracture was performed to determine the mechanical properties of the proximal tibia. Urine was collected on days 0, 14, 28, 42 and 56 and serum on days 0, 28 and 56 to assess biochemical markers of bone turnover including deoxypyridinoline crosslinks and osteocalcin. Both biochemical markers of bone turnover were analyzed by ELISA. Trabecular bone mass, structure, and connectivity density in the Cenestin-treated groups did not differ statistically ( p>0.05) from those of the sham-operated + vehicle-treated rats, but all were significantly higher ( p<0.05) than in the ovx + vehicle-treated rats. Structure Model Index, a measure of trabecular plate morphometry, in Cenestin-treated rats maintained a more equal mix of plate- and rod-like structures similar to the sham group, whereas the ovx group had predominantly rod-like trabeculae. Fracture load in the Cenestin (16.24 mg/kg) treated group was 31% ( p<0.01) higher than in the sham + vehicle-treated group and 61% ( p<0.05) higher than in the ovx + vehicle-treated group. Both the sham-operated + vehicle-treated and Cenestin-treated groups showed significantly lower urinary deoxypyridinoline crosslink excretion at all timepoints and serum osteocalcin at day 56 compared with the ovx + vehicle-treated group. In summary, Cenestin maintained trabecular bone microarchitecture and strength in an ovariectomized rat model of estrogen deficiency.

Osteoporosis is a disease of excess bone fragility that results from both the loss of bone mass and trabecular bone microarchitecture, thereby creating a very fragile skeleton. The purpose of this study was to determine whether treatment of ovariectomized (OVX) osteopenic rats with basic fibroblast growth factor (bFGF) would stimulate the production of new trabeculae, and whether the newly formed trabeculae would make physical connections with the pre-existing trabeculae after prolonged estrogen deficiency. Six-month-old Sprague Dawley rats were OVXed or sham-operated and were left untreated until day 60 post-OVX. A high resolution microscopic scan (XTM) of the right proximal tibia was performed on groups 1 and 2 on day 1 post-OVX, and was repeated in all animals on day 60 post-OVX. At day 60 groups 1 and 2 were treated with vehicle and groups 3 to 6 were injected with bFGF 200 microg/kg/d intravenously for 15 days. At day 82, all animals obtained another in vivo XTM scan of the right tibia; then group 4 were treated with 17B estradiol 10 microg/kg/3x a week, group 5 were treated with hPTH (1-34) at 80 microg/kg/d for 35 days, group 6 were sacrificed, and groups 1 and 2 were treated with vehicle injections for 35 days. At day 110, all remaining animals were sacrificed, and repeat ex vivo XTM scans of the right proximal tibia were performed. Trabecular bone structural variables-including trabecular bone volume, connectivity, number, and thickness-were obtained from all XTM scans. Biochemical markers of bone turnover were also obtained 24 hours before each XTM scan (osteocalcin and deoxypyridinoline), and analyzed by ELISA. Animals OVXed and treated with vehicle had decreased trabecular bone volume, connectivity and number compared to sham-operated animals at both day 60 and day 110. Animals treated with bFGF from day 60-75 post-OVX had evidence of new trabeculae that physically connected with pre-existing trabeculae and also of increased trabecular bone volume seven days after the injections were discontinued. Biochemical markers of bone formation had a small and insignificant increase over baseline levels during the bFGF injections. Bone resorption markers were significantly reduced during the injection period, but returned to baseline levels after the injections were stopped. In addition, we also demonstrated that these newly formed trabecular connections could be maintained or added to with either estrogen or hPTH (1-34) treatments. Thirty-five days after ending the bFGF treatment, trabecular bone volume and connectivity was 25-80% higher in the estrogen and hPTH (1-34) treated animals compared to the untreated animals ( p<0.01). These results support continued development of bFGF as a potential treatment for severely osteoporotic individuals.