Long-term risedronate treatment normalizes mineralization and continues to preserve trabecular architecture: sequential triple biopsy studies with micro-computed tomography.

The objective of the study was to assess the time course of changes in bone mineralization and architecture using sequential triple biopsies from women with postmenopausal osteoporosis (PMO) who received long-term treatment with risedronate. Transiliac biopsies were obtained from the same subjects (n = 7) at baseline and after 3 and 5 years of treatment with 5 mg daily risedronate. Mineralization was measured using 3-dimensional (3D) micro-computed tomography (CT) with synchrotron radiation and was compared to levels in healthy premenopausal women (n = 12). Compared to the untreated PMO women at baseline, the premenopausal women had higher average mineralization (Avg-MIN) and peak mineralization (Peak-MIN) by 5.8% (P = 0.003) and 8.0% (P = 0.003), respectively, and lower ratio of low to high-mineralized bone volume (BMR-V) and surface area (BMR-S) by 73.3% (P = 0.005) and 61.7% (P = 0.003), respectively. Relative to baseline, 3 years of risedronate treatment significantly increased Avg-MIN (4.9 +/- 1.1%, P = 0.016) and Peak-MIN (6.2 +/- 1.5%, P = 0.016), and significantly decreased BMR-V (-68.4 +/- 7.3%, P = 0.016) and BMR-S (-50.2 +/- 5.7%, P = 0.016) in the PMO women. The changes were maintained at the same level when treatment was continued up to 5 years. These results are consistent with the significant reduction of turnover observed after 3 years of treatment and which was similarly maintained through 5 years of treatment. Risedronate restored the degree of mineralization and the ratios of low- to high-mineralized bone to premenopausal levels after 3 years of treatment, suggesting that treatment reduced bone turnover in PMO women to healthy premenopausal levels. Conventional micro-CT analysis further demonstrated that bone volume (BV/TV) and trabecular architecture did not change from baseline up to 5 years of treatment, suggesting that risedronate provided long-term preservation of trabecular architecture in the PMO women. Overall, risedronate provided sustained benefits on mineralization and architecture, two key determinants of bone strength, over 5 years lending support for its long-term efficacy in fracture risk reduction.

Quantitative imaging of proteoglycan in cartilage using a gadolinium probe and microCT.

OBJECTIVE: Micro-computed tomography (microCT) imaging has the potential to allow the three-dimensional (3D) visualization of cartilage morphology. However, cartilage intensity on a microCT image is weak because cartilage does not strongly attenuate X-rays. This work was designed to demonstrate that exposure of cartilage to charged gadolinium compounds modifies the intensity to allow an improved visualization of cartilage morphology and the determination of proteoglycan content. DESIGN: Trypsin was used to deplete proteoglycan in bovine nasal cartilage disks. Disks were then exposed to Gd(3+), gadopentetate (Gd-DTPA(2-)), or gadoteridol (Gd-HP-DO3A), and imaged with microCT. The intensities of the disks were measured from the images and compared to the actual proteoglycan content determined with a dimethylmethylene blue assay. RESULTS: Treatment of naïve disks with 200 mM Gd(3+) for 24h at room temperature produced a 2.8-fold increase in intensity on microCT images. Similar treatment with 200 mM Gd-DTPA(2-) produced a 1.4-fold increase. After 2h of trypsin treatment at room temperature, the intensities of cartilage disks exposed to 20 0mM Gd(3+) decreased by 12%. Conversely, the intensities of trypsin-treated disks exposed to 200 mM Gd-DPTA(2-) increased by 15%. Trypsin treatment caused a 4% increase in the intensities of disks exposed to neutral Gd-HP-DO3A. The correlation between proteoglycan content and the microCT intensity of cartilage treated with Gd(3+) was very good (r(2)=0.81). CONCLUSIONS: Gadolinium and microCT allow an improved 3D visualization of cartilage and quantification of its proteoglycan content.

Risedronate preserves bone architecture in early postmenopausal women in 1 year as measured by three-dimensional microcomputed tomography.

Risedronate reduces the risk of vertebral fractures by up to 70% within the first year of treatment. Increases in bone mineral density or decreases in bone turnover markers explain only a portion of the anti-fracture effect, suggesting that other factors, such as changes in trabecular bone architecture, also play a role. Our objective was to determine the effects of risedronate on bone architecture by analyzing iliac crest bone biopsy specimens using three-dimensional microcomputed tomography (3-D micro CT). Biopsy specimens were obtained at baseline and after 1 year of treatment from women enrolled in a double-blind, placebo-controlled study of risedronate 5 mg daily for the prevention of early postmenopausal bone loss. Trabecular architecture deteriorated in the placebo group (n = 12), as indicated by a 20.3% decrease in bone volume (25.1% vs. 20.0%, P = 0.034), a 13.5% decrease in trabecular number (1.649 vs. 1.426 mm(-1), P = 0.052), a 13.1% increase in trabecular separation (605 vs. 684 microm, P = 0.056), and an 86.2% increase in marrow star volume (3.251 vs. 6.053 mm(3), P = 0.040) compared with baseline values. These changes in architectural parameters occurred in the presence of a concomitant decrease from baseline in lumbar spine bone mineral density (-3.3%, P = 0.002), as measured by dual energy x-ray absorptiometry. There was no statistically significant ( P < 0.05) deterioration in the risedronate-treated group (n = 14) over the 1-year treatment period. Comparing the actual changes between the two groups, the placebo group experienced decreases in bone volume (placebo, -5.1%; risedronate, +3.5%; P = 0.011), trabecular thickness (placebo, -20 microm; risedronate, +23 microm; P = 0.032), and trabecular number (placebo, -0.223 mm(-1); risedronate, +0.099 mm(-1); P = 0.010), and increases in percent plate (placebo, +2.79%; risedronate, -3.23%; P = 0.018), trabecular separation (placebo, +79 microm; risedronate, -46 microm; P = 0.010) and marrow star volume (placebo, +2.80 mm(3); risedronate, -2.08mm(3); P = 0.036), compared with the risedronate group. These data demonstrate that trabecular architecture deteriorated significantly in this cohort of early postmenopausal women, and that this deterioration was prevented by risedronate. Although there is no direct link in this study between fracture and preservation of architecture, it is reasonable to infer that the preservation of bone architecture may play a role in risedronate's anti-fracture efficacy.

Three-dimensional microimaging (MRmicroI and microCT), finite element modeling, and rapid prototyping provide unique insights into bone architecture in osteoporosis.

With the proportion of elderly people increasing in many countries, osteoporosis has become a growing public health problem, with rising medical, social, and economic consequences. It is well recognized that a combination of low bone mass and the deterioration of the trabecular architecture underlies osteoporotic fractures. A comprehensive understanding of the relationships between bone mass, the three-dimensional (3D) architecture of bone and bone function is fundamental to the study of new and existing therapies for osteoporosis. Detailed analysis of 3D trabecular architecture, using high-resolution digital imaging techniques such as magnetic resonance microimaging (MRmicroI), micro-computed tomography (microCT), and direct image analysis, has become feasible only recently. Rapid prototyping technology is used to replicate the complex trabecular architecture on a macroscopic scale for visual or biomechanical analysis. Further, a complete set of 3D image data provides a basis for finite element modeling (FEM) to predict mechanical properties. The goal of this paper is to describe how we can integrate three-dimensional microimaging and image analysis techniques for quantitation of trabecular bone architecture, FEM for virtual biomechanics, and rapid prototyping for enhanced visualization. The integration of these techniques provide us with an unique ability to investigate the role of bone architecture in osteoporotic fractures and to support the development of new therapies.

Evaluation of changes in trabecular bone architecture and mechanical properties of minipig vertebrae by three-dimensional magnetic resonance microimaging and finite element modeling.

The study objective was to analyze the three-dimensional (3D) trabecular architecture and mechanical properties in vertebral specimens of young and mature Sinclair minipigs to assess the relative contribution of architecture to bone strength. We used 3D magnetic resonance microimaging (MRmicroI) and direct image analysis to evaluate a set of standard structural measurements and new architectural descriptors of trabecular bone in biopsy specimens from L2, L3, and L4 vertebrae (n = 16 in each group) from young (mean age, 1.2 years) and mature (mean age, 4.8 years) minipigs. The measurements included bone volume/tissue volume (BV/TV), marrow star volume (Ma.St.V), connectivity density (ConnD), and two new parameters, percent platelike trabeculae (% plate) and percent bone in the load direction (% boneLD). The % plate, calculated from surface curvature, allowed the delineation of plates from rods. The % boneLD quantified the percentage of bone oriented along the long axis of the vertebral body. We showed that 3D MRmicroI can detect the subtle changes in trabecular architecture between the two age groups. ConnD, star volume, % plate, % boneLD, and BV/TV were found to be more effective than the model-based, derived indices (trabecular thickness [Tb.Th], trabecular separation [Tb.Sp], and trabecular number [Tb.N]) in differentiating the structural changes. BV/TV, % plate, and % boneLD significantly increased (p < 0.05) in all three vertebral sites of the mature minipigs. The significant decrease in ConnD and star volume in the mature vertebra was consistent with the concurrent increase of platelike trabecular bone (p < 0.05). Overall, ConnD, star volume, % plate, and % boneLD provided a coherent picture of the architectural changes between the two age groups. Apparent modulus and maximum stress were determined experimentally on biopsy specimens from L2 vertebrae (n = 16). When apparent modulus was predicted using 3D MRmicroI data sets as input for finite element modeling (FEM), the results were similar to the experimentally determined apparent modulus (p = 0.12). Both methods were then used to compare the young and the mature animals; the experimental and predicted apparent modulus were significantly higher for the mature group (p = 0.003 and 0.012, respectively). The experimental maximum stress in the vertebra of the mature animals was twice as high as that for the young animals (p = 0.006). Bone quantity (BV/TV or bone mineral content [BMC]) alone could explain approximately 74-85% of the total variability in stress and modulus. The inclusion of either ConnD or % boneLD with BV/TV in a multiple regression analysis significantly improved the predictability of maximum stress, indicating that architecture makes additional contributions to compressive strength in normal minipig vertebra.

Bone architecture and image synthesis.

3-D bone architecture can now be measured by micro-computer tomography or micro-magnetic resonance imaging. The principles of the micro-computer technique is reviewed and new architectural parameters can be computed. In addition, the method allows the contruction of polymer models by stereolithography, a method that can be used to perform repetitive mechanical studies on the same bone sample. These non destructive methods are interesting in the pre-clinical studies on bone diseases and in the investigation of animal trials on new pharmacological compounds active on bone.

Advances in automated 3-D image analyses of cell populations imaged by confocal microscopy.

Automated three-dimensional (3-D) image analysis methods are presented for rapid and effective analysis of populations of fluorescently labeled cells or nuclei in thick tissue sections that have been imaged three dimensionally using a confocal microscope. The methods presented here greatly improve upon our earlier work (Roysam et al.:J Microsc 173: 115-126, 1994). The principal advances reported are: algorithms for efficient data pre-processing and adaptive segmentation, effective handling of image anisotrophy, and fast 3-D morphological algorithms for separating overlapping or connected clusters utilizing image gradient information whenever available. A particular feature of this method is its ability to separate densely packed and connected clusters of cell nuclei. Some of the challenges overcome in this work include the efficient and effective handling of imaging noise, anisotrophy, and large variations in image parameters such as intensity, object size, and shape. The method is able to handle significant inter-cell, intra-cell, inter-image, and intra-image variations. Studies indicate that this method is rapid, robust, and adaptable. Examples were presented to illustrate the applicability of this approach to analyzing images of nuclei from densely packed regions in thick sections of rat liver, and brain that were labeled with a fluorescent Schiff reagent.

A gray-level thinning method for delineation and representation of arteries.

The quantification of three dimensional (3D) properties of coronary arteries is of significant importance. The performance of the 3D analysis is critically based on low-level representation of the arterial tree for different projections. A skeletal representation of arteries can provide appropriate data structure for registration of multiple angiographic projections and it can be further utilized for 3D reconstruction of the arterial tree. This paper presents an automated method for extracting the skeletal points of an arterial tree directly from the gray-level information without determining the edges a priori. It offers the advantage of improved reliability compared to methods based on detecting dual edges of the arteries. Novel application of filtering techniques provide accurate estimates of the statistics of the background. A recursive search scheme is used to aggregate the skeletal representation at multiple resolutions. Results on a set of Digitally Subtracted Angiograms (DSA) have been presented.