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The use of micro-CT to study bone architecture dynamics noninvasively
Drug Discovery Today: Technologies
Vol. 3, No. 2 2006
Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Technologies for whole animal pharmacology
The use of micro-CT to study bone architecture dynamics noninvasively Jacqueline C. van der Linden, Jan H. Waarsing, Harrie Weinans* Department Orthopaedics, Erasmus MC, Rotterdam, The Netherlands
High-resolution micro-CT has become a standard tool in the evaluation of bone architecture. It has recently progressed from an invasive tool for bone specimens into an in vivo tool for small animals. The combination
Section Editors: Bart Ellenbroek – Radboud University, Nijmegen, The Netherlands Twan Ederveen – N.V. Organon, Oss, The Netherlands
of novel sophisticated evaluation methods, such as registration (matching) of sequential scans and computer simulation models will further evolve in vivo micro-CT into an optimal tool for small animal phenotyping and contemporary approaches for drug discovery relating to the skeleton. Introduction X-ray computed tomography (CT) was introduced in the 1970s and quickly developed into a clinical tool. The downscaling of this technique to resolutions below 100 mm took another 10– 15 years and only recently the technique matured to provide commercially available table-top devices enabling high-resolution scans of small specimens. Some investigators used synchrotron radiation, whose monochromatic parallel X-ray beams dramatically improved the quality and resolution of the images [1,2]. Ford Motor Companies (Detroit) significantly contributed to the development of micro-CT by building a system with a superior X-ray tube and an image intensifier detector. In addition, they developed the appropriate software (a cone beam reconstruction algorithm [3]) that allowed the creation of a three-dimensional (3D) array of data instead of a series of two-dimensional (2D) slices. This not only sped up the 3D reconstruction, but also provided a resolution in the order of 50 mm both in plane and in slice thickness. In particular, the latter aspect provided a dramatic improvement upon existing *Corresponding author: H. Weinans ([email protected]) 1740-6749/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2006.06.006
technologies and was quickly applied in the medical field by bone scientists in the Detroit area [4,5]. It took an effort to improve upon this standard. Several groups built similar dedicated devices with resolutions below 100 mm [6–8]. In the 1990s, this resulted in a few commercial micro-CT table-top devices and a boost of publications using micro-CT in medical applications, in particular with respect to bone research. Currently, the challenge is to develop more sophisticated postprocessing techniques for the 3D micro-CT data and to further the technology to develop an optimal in vivo evaluation system with low radiation, high resolution and short scanning times. These combined efforts on improving the technology should allow multiple, sequential scans in small animal experiments at an improved resolution resulting in higher quality of the (postprocessed) data. This should finally bring us an optimized technology for phenotyping and studying pathologies in small animals, thereby providing supreme opportunities in skeletal drug discovery.
Micro-CT applications The obvious initial advantage of micro-CT was that changes in bone architecture could be studied in 3D. This was thought to yield more valuable information than the traditional methods, which used 2D sections of bone. New methods and computer algorithms were developed to characterize the 3D architecture. An important goal of introducing 3D morphometry measures of cancellous bone was to identify those morphometric 213
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parameters that correlated with other more clinically related parameters of bone, such as mechanical strength, stiffness or the fracture risk of a patient. In part, the new micro-CT-based parameters were 3D versions of architectural parameters, which were already used in 2D bone histomorphometry [9]. In 2D histomorphometry, 3D-model assumptions were required to derive parameters such as trabecular thickness, trabecular spacing and trabecular number [10], whereas the 3D versions of these parameters could now be obtained in an unbiased manner because the 3D architecture was known. Besides this, new measures were developed, such as connectivity density, degree of anisotropy [11] and structure model index [12], which is a quantitative measure that indicates whether the shape of the trabeculae is rod-like or plate-like. It was known for a long time that bone mineral density (BMD), or its related parameter bone volume fraction, cannot fully explain the mechanical characteristics of cancellous bone and has severe shortcomings in predicting fracture risk. The new parameters aimed to reduce this shortcoming by adding information that is independent of BMD. This information could then become a target for drug testing, providing measures that are at least as reliable as BMD, preferably more so. It is clear that 3D morphometry analyses from micro-CT taught us a lot about bone quality in general and specifically the crucial role of bone architecture for its mechanical properties [13–15]. However, parameters from 3D morphometry that can be used as a surrogate for bone quality or fracture risk and are a drastic improvement on BMD have not yet emerged. The most relevant parameter in this respect is probably the orientation of the trabeculae or the degree of anisotropy, which seems to contribute to the estimation of fracture risk independently of BMD [16,17].
In silico testing Other novel applications of micro-CT are the use of computer models or in silico testing of the strength and stiffness of cancellous bone samples. Many skeletal diseases that affect the bone tissue result in a weaker skeleton and a reduced strength or stiffness of the bones as a whole. This deterioration of properties can be caused essentially by changes in two physical properties of bone: (i) the micro-architecture and (ii) the quality of the mineralized bone tissue. By combining micro-CT scans, finite element computer modeling and mechanical testing, it is possible to differentiate between the above properties ([18], Fig. 1). This method is very useful in investigating the detailed effects of pharmacological treatment. For example, Day et al. [19] used vertebral bone specimens of dogs treated with high-dose bisphosphonates to investigate how this drug strengthens cancellous bone, and whether the accumulation of microdamage that occurred in bisphosphonate-treated animals would affect the mechanical properties of their bones. These authors were able to use a combination of micro-CT, finite element modeling and 214
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mechanical testing to examine the treatment effects on the calcified matrix and trabecular architecture independently (Fig. 1). The increased stiffness of the whole specimen as a consequence of bisphosphonate treatment could be fully explained from the small increase in BMD combined with changes in architecture (Fig. 1). The analyses showed that tissue stiffness had not changed. However, the density of the tissue (the calcified matrix) measured from an Archimedes test was clearly increased in the drug-treated specimens. This higher tissue density is probably related to a higher tissue mineralization in the bisphosphonate-treated bones, which should normally result in increased tissue stiffness. Some authors even suggest that this secondary mineralization is part of the working mechanism of bisphosphonates [20]. In the dog study of Day et al. [19], however, the increase in stiffness provided by higher tissue density of the trabeculae itself was probably counteracted by the increase in microdamage, which was also detected in the drug-treated specimens using histological techniques [21]. With traditional histomorphometric techniques, changes in bone remodeling can be detected: changes in osteoblastic bone formation and osteoclastic bone resorption can be quantified [10]. Although micro-CT is not capable of providing such information, it can be used for in silico computer simulations of the bone resorption and bone formation process. Pharmacological agents that are used to treat osteoporosis, such as bisphosphonates, calcitonin or selective estrogen receptor modulators (SERMs), reduce bone resorption and slow down the bone remodeling process. As a result, bone loss is slowed down or bone mass is even increased, resulting in a decreased fracture risk. Van der Linden et al. [15] used a 3D model of human trabecular vertebral bone to simulate the bone remodeling process: the formation of cavities and refill of these cavities (Fig. 2). Using this model, the effects of changes in bone remodeling caused by antiresorptive treatment were investigated. Such detailed studies provided more insight into the importance of the size and extension of the resorption pits in the bone remodeling process. Although the modeling of small specimens, such as cylinders or cubes of trabecular bone, gives valuable information on bone architecture and tissue stiffness, both the trabecular bone and the cortex are normally affected in bone diseases. Therefore, it is interesting to look at the mechanical behaviour of the bone as a whole rather than of small bone specimens. With increasing memory and computing power, it became possible to simulate the loading of a whole femur using a supercomputer. Van Rietbergen et al. [22] performed a study in which they investigated the load distribution in a healthy and an osteoporotic femur. The bones were scanned by micro-CT to obtain a 3D model of the whole femur. Subsequently, boundary conditions were applied to the bone to simulate normal loading and the strain distribution within the bones was calculated.
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Drug Discovery Today: Technologies | Technologies for whole animal pharmacology
Figure 1. (a) The stiffness of the mineralized tissue of the bone (the tissue of which the trabeculae are made or sometimes called bulk stiffness) can be determined by combining real mechanical tests with computer simulations. The mechanical compression tests in three directions (anteroposterior [AP], mediolateral [ML] and craniocaudal [CC]) yield stress–strain curves, the slopes of these curves yield the stiffness of the whole specimen (i.e. the apparent stiffness). Using a micro-CT scanner, a computer model of the same specimen is made to simulate the same compression test in a finite element simulation (top part). A fit can be made between experiment and computer model by choosing the value for the stiffness of the tissue of the mineralized trabeculae. (b) The graphs show the similarity between the overall stiffness of the specimen in experiments performed with dogs treated with two types of bisphosphonate (risedronate and alendronate) and untreated controls. The physical tests were compared with a computer model. One value for the tissue stiffness matched all the experiments for both bisphosphonates and control. Hence, the computer model is an excellent simulation of the experiment and the tissue stiffness required for the fit was the same in every treatment protocols [19].
This study showed differences in load distribution between healthy and osteoporotic bone. In the osteoporotic bone, the average strain was higher. Furthermore, the strain distribution was wider, indicating that in the osteoporotic bone more trabeculae showed either very small or large loads compared to the healthy bone. The available bone in the femur was used less efficiently in the osteoporotic femur than in the healthy femur.
In vivo micro-CT Conventional micro-CT has to be considered an invasive measurement method owing to the high X-ray radiation dose needed to obtain good-quality image data, and thus studies
utilizing the method have generally been cross-sectional. To be able to visualize longitudinal changes in bone mass in vivo, many studies have used peripheral quantitative CT (pQCT) [23,24]. The low resolutions, especially in slice thickness, used in this modality made it possible to perform in vivo imaging in small animals. Although the resolution inside a cross-section (in the order of 100 mm) is high enough to distinguish cortical and trabecular bone and to assess trabecular bone mass, the resolution is too low to resolve the trabecular architecture. The first in vivo high-resolution (<50 mm 3D) micro-CT images were made of rat tibiae using synchrotron radiation [2]. Advances have been made in the development of table-top micro-CT devices through the www.drugdiscoverytoday.com
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Figure 2. Simulating the bone renewal (remodeling) process in three dimensions using models created from micro-CT scans. The formation of cavities and refill of these cavities are simulated: resorption cavities are created with a random distribution over the surface of the trabeculae. Trabeculae that are disconnected are not repaired, all cavities that do not disconnect trabeculae are refilled. The remodeling parameters (number of resorption cavities, resorption depth and formation deficit) were based on histological data from the literature. From the model, it was concluded that most of the bone loss with aging is due to the formation deficit (the negative bone balance in each remodeling unit: the amount of bone made by osteoblasts is normally slightly smaller than the amount of bone resorbed by osteoclasts [69–95%]), followed by bone loss because of breached trabeculae that were not repaired (1–21%).
introduction of rotating source-detector systems, in which the animal is placed in a fixed bed, similar to that used for clinical CT systems. Moreover, as both computing memory and the resolution of charge-coupled device (CCD) cameras increased, the voxel size that could be achieved using microCT scanners decreased. Current high-end scanners use up to 10 Megapixel cameras with voxel sizes below 5 mm. These technological improvements have enabled the development of table-top in vivo micro-CT scanners with resolutions in the order of 10–50 mm and radiation dosages below 1 Gy per scan [8,25,26]. Follow-up studies using such devices showed halted bone growth and loss of trabecular bone in hind-limb suspended rats in contrast to trabecular reorganization in normally aging rats [25], and revealed the similarity in the bone loss dynamics of rats after ovariectomy and normally aging rats [26,27]. Another study showed the feasibility of using in vivo micro-CT to detect the increase in trabecular thickness caused by treatment with parathyroid hormone (PTH) and the bone-protective effect of various antiresorptive agents [28]. Also, bone resorption and adaptation in the rat fibula after osteotomy have been monitored successfully in vivo [29], as have the subchondral bone changes occurring in a rat model of osteoarthritis [30]. One of the biggest advantages of in vivo imaging, as concluded in most of the above-mentioned studies, is the 216
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increase in statistical power, because treatment effects can be measured independently of biological base-line variations. Thus, it is possible to detect smaller effects in an earlier stage than is possible with cross-sectional imaging modalities, such as conventional micro-CT and histology. Related to this, fewer animals are needed to perform an experiment, which can reduce both the cost and the animal inconvenience of each experiment.
In vivo bone architecture dynamics The possibilities of in vivo micro-CT are increased further when image-registration techniques are used [26,27,29]. Image registration comprises a set of image processing techniques that are generally used to match, for example, clinical CT images from longitudinal studies or to match images obtained from different imaging modalities like CT, magnetic resonance imaging (MRI) and positron emission tomography (PET) or single photon emission computed tomography (SPECT) [30–32]. In short, these techniques iteratively reposition one scanned set of images with respect to another set, until a certain mathematical criterion is optimized and the two sets ‘match’ optimally. When applied to longitudinal scans obtained by in vivo micro-CT, two datasets of a bone of an individual animal can be matched such that they can be positioned on top of each other. Thus, it becomes possible to
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visualize and quantify where new bone is formed, and where old bone was resorbed ([26], Figs 3 and 4). This contrasts with traditional dynamic histomorphometry, which can only be used to measure new bone formation, obviously ex vivo. These registered images have also been used to observe the result of bone remodeling dynamics on single trabeculae [27]. It could be shown in a rat model of postmenopausal bone loss not only that are trabeculae resorbed but that the remaining trabeculae increase in thickness by apposition of new bone, and that several connected trabeculae remodel into thick trabecular struts (Fig. 5). Further, such longitudinal 3D datasets could be used to locally quantify changes in thickness (Fig. 6). The latter possibilities indicate the potential of in vivo micro-CT for drug-related research. Because the current paradigm in bone research is bone quality, which includes among
Figure 3. 3D longitudinal slab from the proximal tibia of an ovariectomized rat. Owing to ovariectomy there will be a reduction in estrogen levels and a subsequent loss of bone. This bone loss is often shown in the proximal femur of ovariectomized rats as a model for human osteoporosis. The figure shows the locations of bone resorption (yellow) and formation of new bone (red) in the first 4 weeks after ovariectomy. Clearly, the overall effects of ovariectomy are cancellous bone loss (mid-metphyseal part), but there are certain locations with bone apposition (e.g. the medial endo-cortex). These detailed analyses can elucidate the precise effects of different drugs that are under development or help to better understand existing drugs for osteoporosis.
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Figure 4. Fluorescent labeling confirms local sites of bone formation as suggested by in vivo micro-CT. (a) An histological section with a green fluorescent label that is incorporated at locations of new bone formation and (b) an in vivo measurement showing the same locations of bone formation and showing bone resorption locations in red and bone apposition in yellow that matches the green label in the histological section [26].
others bone architecture, there is a need to know how drugs that are used to influence bone metabolism affect trabecular architecture. Take, for instance, the ongoing debate on the bone anabolic effect of PTH treatment. It is generally accepted that PTH treatment can lead to the apposition of new bone on existing surfaces, but the debate focuses on the question of whether new bone struts can be formed that connect previously unconnected trabeculae. Although such actions have never been shown to really exist, recent in vivo micro-CT findings did show that bone remodeling is very dynamic and can transform existing trabecular structures into completely different structures. There is a need for caution when using in vivo micro-CT because of the influence of ionizing X-ray radiation on bone metabolism. Published studies report radiation doses of between 0.2 and 1.0 Gy per scan. Although relatively low, such a dose is still about 10,000 times greater than that generated by a general chest X-ray. A single dose as low as 0.25 Gy negatively affects bone marrow in humans [33] and thus caution is justified. Radiation doses below 1 Gy have been shown to affect rodent metabolism [34,35], although no effects of such doses have been found in bones and the epiphyseal growth-plates [36,37]. These studies indicate that a single dose of around 0.5 Gy is not likely to affect bone metabolism measurably. However, longitudinal studies involve multiple scans on the same animal, which increase radiation damage dependent on the number of the scans and the amount of time between the scanning sessions. Multiple scans of aged female wistar rats did not result in detectable differences in trabecular architecture [27,38]. In mice, however, multiple scans did result in detectable, although minor, www.drugdiscoverytoday.com
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Figure 5. Registered cross-sections from the tibial metaphysis of a rat. The subsequent in vivo measurements show the dynamics of bone remodeling as the rat ages from 1- to 2-year-old. Notice how remaining trabeculae increase in thickness and the remodeling of several thin trabeculae into one big rod.
bone loss in radiated legs relative to nonradiated contralateral legs [38,39]. The radio-sensitivity of the bones of experimental animals appears to depend on species, strains, age and bone site. In general, active proliferating cells are most sensitive to radiation, thus extra care needs to be taken with animals that have an active growth-plate and high remodeling rates.
Figure 6. Visualisation of local trabecular thickness in a 3D sample of metaphyseal trabecular bone in an ovariectomized rat at two time points (0 and 4 weeks after ovariectomy), and a visualization of the changes in thickness presented in the 4 week scan. Bone that was not present at week 0 (thus newly formed bone) is visualized transparent. Bone that remains becomes thicker (on average). The blue parts become thinner and will probably be removed in the next stage of the bone loss process.
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Conclusions In summary, micro-CT analysis has proven its value for bone morphometry and the estimation of bone stiffness and strength when it is combined with finite element computer models. In addition, the 3D reconstructions from micro-CT enable computer simulations of bone turnover that contribute to the understanding of the mechanisms of the bone metabolic process and its relation with mechanical parameters. Recent introduction of in vivo application of microCT showed the dynamic activities of single trabeculae and could in principle lead to a breakthrough for fast and accurate evaluation of drugs that effect bone metabolism. In particular for studies involving osteoporosis and osteoarthritis such an assessment is extremely useful. Progress can still be made with respect to decreasing the radiation dose. In particular, improvements in (micro-)CT reconstruction algorithms can be made. Such solutions can potentially improve the quality of the reconstructed data set especially when the images have a poor signal to noise ratio and thus allow shorter scanning time, similar to approaches in SPECT applications [40]. At the same time, there seems to be some progress in the development of monochromatic Xray beams, which might dramatically improve future X-ray (micro-)CT applications as well [41]. These combined novel software and hardware approaches might lead to new breakthroughs in micro-CT imaging, in particular for in vivo applications. Further developments in postprocessing software that interacts with micro-CT will provide more accurate information from the 3D images and can lead to highthroughput devices for fast phenotyping of small animals, which will be of great value in contemporary bone disease and other drug discovery programs.
Acknowledgements J.C.v.d.L. was supported by STW (Veni-program, RPG 6294) and J.H.W. was supported by the Dutch Arthritis Foundation. Part of the work presented was supported by the EU (grant QLRT-1999-02024, MIAB).
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Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Technologies for whole animal pharmacology
The use of micro-CT to study bone architecture dynamics noninvasively Jacqueline C. van der Linden, Jan H. Waarsing, Harrie Weinans* Department Orthopaedics, Erasmus MC, Rotterdam, The Netherlands
High-resolution micro-CT has become a standard tool in the evaluation of bone architecture. It has recently progressed from an invasive tool for bone specimens into an in vivo tool for small animals. The combination
Section Editors: Bart Ellenbroek – Radboud University, Nijmegen, The Netherlands Twan Ederveen – N.V. Organon, Oss, The Netherlands
of novel sophisticated evaluation methods, such as registration (matching) of sequential scans and computer simulation models will further evolve in vivo micro-CT into an optimal tool for small animal phenotyping and contemporary approaches for drug discovery relating to the skeleton. Introduction X-ray computed tomography (CT) was introduced in the 1970s and quickly developed into a clinical tool. The downscaling of this technique to resolutions below 100 mm took another 10– 15 years and only recently the technique matured to provide commercially available table-top devices enabling high-resolution scans of small specimens. Some investigators used synchrotron radiation, whose monochromatic parallel X-ray beams dramatically improved the quality and resolution of the images [1,2]. Ford Motor Companies (Detroit) significantly contributed to the development of micro-CT by building a system with a superior X-ray tube and an image intensifier detector. In addition, they developed the appropriate software (a cone beam reconstruction algorithm [3]) that allowed the creation of a three-dimensional (3D) array of data instead of a series of two-dimensional (2D) slices. This not only sped up the 3D reconstruction, but also provided a resolution in the order of 50 mm both in plane and in slice thickness. In particular, the latter aspect provided a dramatic improvement upon existing *Corresponding author: H. Weinans ([email protected]) 1740-6749/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2006.06.006
technologies and was quickly applied in the medical field by bone scientists in the Detroit area [4,5]. It took an effort to improve upon this standard. Several groups built similar dedicated devices with resolutions below 100 mm [6–8]. In the 1990s, this resulted in a few commercial micro-CT table-top devices and a boost of publications using micro-CT in medical applications, in particular with respect to bone research. Currently, the challenge is to develop more sophisticated postprocessing techniques for the 3D micro-CT data and to further the technology to develop an optimal in vivo evaluation system with low radiation, high resolution and short scanning times. These combined efforts on improving the technology should allow multiple, sequential scans in small animal experiments at an improved resolution resulting in higher quality of the (postprocessed) data. This should finally bring us an optimized technology for phenotyping and studying pathologies in small animals, thereby providing supreme opportunities in skeletal drug discovery.
Micro-CT applications The obvious initial advantage of micro-CT was that changes in bone architecture could be studied in 3D. This was thought to yield more valuable information than the traditional methods, which used 2D sections of bone. New methods and computer algorithms were developed to characterize the 3D architecture. An important goal of introducing 3D morphometry measures of cancellous bone was to identify those morphometric 213
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parameters that correlated with other more clinically related parameters of bone, such as mechanical strength, stiffness or the fracture risk of a patient. In part, the new micro-CT-based parameters were 3D versions of architectural parameters, which were already used in 2D bone histomorphometry [9]. In 2D histomorphometry, 3D-model assumptions were required to derive parameters such as trabecular thickness, trabecular spacing and trabecular number [10], whereas the 3D versions of these parameters could now be obtained in an unbiased manner because the 3D architecture was known. Besides this, new measures were developed, such as connectivity density, degree of anisotropy [11] and structure model index [12], which is a quantitative measure that indicates whether the shape of the trabeculae is rod-like or plate-like. It was known for a long time that bone mineral density (BMD), or its related parameter bone volume fraction, cannot fully explain the mechanical characteristics of cancellous bone and has severe shortcomings in predicting fracture risk. The new parameters aimed to reduce this shortcoming by adding information that is independent of BMD. This information could then become a target for drug testing, providing measures that are at least as reliable as BMD, preferably more so. It is clear that 3D morphometry analyses from micro-CT taught us a lot about bone quality in general and specifically the crucial role of bone architecture for its mechanical properties [13–15]. However, parameters from 3D morphometry that can be used as a surrogate for bone quality or fracture risk and are a drastic improvement on BMD have not yet emerged. The most relevant parameter in this respect is probably the orientation of the trabeculae or the degree of anisotropy, which seems to contribute to the estimation of fracture risk independently of BMD [16,17].
In silico testing Other novel applications of micro-CT are the use of computer models or in silico testing of the strength and stiffness of cancellous bone samples. Many skeletal diseases that affect the bone tissue result in a weaker skeleton and a reduced strength or stiffness of the bones as a whole. This deterioration of properties can be caused essentially by changes in two physical properties of bone: (i) the micro-architecture and (ii) the quality of the mineralized bone tissue. By combining micro-CT scans, finite element computer modeling and mechanical testing, it is possible to differentiate between the above properties ([18], Fig. 1). This method is very useful in investigating the detailed effects of pharmacological treatment. For example, Day et al. [19] used vertebral bone specimens of dogs treated with high-dose bisphosphonates to investigate how this drug strengthens cancellous bone, and whether the accumulation of microdamage that occurred in bisphosphonate-treated animals would affect the mechanical properties of their bones. These authors were able to use a combination of micro-CT, finite element modeling and 214
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mechanical testing to examine the treatment effects on the calcified matrix and trabecular architecture independently (Fig. 1). The increased stiffness of the whole specimen as a consequence of bisphosphonate treatment could be fully explained from the small increase in BMD combined with changes in architecture (Fig. 1). The analyses showed that tissue stiffness had not changed. However, the density of the tissue (the calcified matrix) measured from an Archimedes test was clearly increased in the drug-treated specimens. This higher tissue density is probably related to a higher tissue mineralization in the bisphosphonate-treated bones, which should normally result in increased tissue stiffness. Some authors even suggest that this secondary mineralization is part of the working mechanism of bisphosphonates [20]. In the dog study of Day et al. [19], however, the increase in stiffness provided by higher tissue density of the trabeculae itself was probably counteracted by the increase in microdamage, which was also detected in the drug-treated specimens using histological techniques [21]. With traditional histomorphometric techniques, changes in bone remodeling can be detected: changes in osteoblastic bone formation and osteoclastic bone resorption can be quantified [10]. Although micro-CT is not capable of providing such information, it can be used for in silico computer simulations of the bone resorption and bone formation process. Pharmacological agents that are used to treat osteoporosis, such as bisphosphonates, calcitonin or selective estrogen receptor modulators (SERMs), reduce bone resorption and slow down the bone remodeling process. As a result, bone loss is slowed down or bone mass is even increased, resulting in a decreased fracture risk. Van der Linden et al. [15] used a 3D model of human trabecular vertebral bone to simulate the bone remodeling process: the formation of cavities and refill of these cavities (Fig. 2). Using this model, the effects of changes in bone remodeling caused by antiresorptive treatment were investigated. Such detailed studies provided more insight into the importance of the size and extension of the resorption pits in the bone remodeling process. Although the modeling of small specimens, such as cylinders or cubes of trabecular bone, gives valuable information on bone architecture and tissue stiffness, both the trabecular bone and the cortex are normally affected in bone diseases. Therefore, it is interesting to look at the mechanical behaviour of the bone as a whole rather than of small bone specimens. With increasing memory and computing power, it became possible to simulate the loading of a whole femur using a supercomputer. Van Rietbergen et al. [22] performed a study in which they investigated the load distribution in a healthy and an osteoporotic femur. The bones were scanned by micro-CT to obtain a 3D model of the whole femur. Subsequently, boundary conditions were applied to the bone to simulate normal loading and the strain distribution within the bones was calculated.
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Figure 1. (a) The stiffness of the mineralized tissue of the bone (the tissue of which the trabeculae are made or sometimes called bulk stiffness) can be determined by combining real mechanical tests with computer simulations. The mechanical compression tests in three directions (anteroposterior [AP], mediolateral [ML] and craniocaudal [CC]) yield stress–strain curves, the slopes of these curves yield the stiffness of the whole specimen (i.e. the apparent stiffness). Using a micro-CT scanner, a computer model of the same specimen is made to simulate the same compression test in a finite element simulation (top part). A fit can be made between experiment and computer model by choosing the value for the stiffness of the tissue of the mineralized trabeculae. (b) The graphs show the similarity between the overall stiffness of the specimen in experiments performed with dogs treated with two types of bisphosphonate (risedronate and alendronate) and untreated controls. The physical tests were compared with a computer model. One value for the tissue stiffness matched all the experiments for both bisphosphonates and control. Hence, the computer model is an excellent simulation of the experiment and the tissue stiffness required for the fit was the same in every treatment protocols [19].
This study showed differences in load distribution between healthy and osteoporotic bone. In the osteoporotic bone, the average strain was higher. Furthermore, the strain distribution was wider, indicating that in the osteoporotic bone more trabeculae showed either very small or large loads compared to the healthy bone. The available bone in the femur was used less efficiently in the osteoporotic femur than in the healthy femur.
In vivo micro-CT Conventional micro-CT has to be considered an invasive measurement method owing to the high X-ray radiation dose needed to obtain good-quality image data, and thus studies
utilizing the method have generally been cross-sectional. To be able to visualize longitudinal changes in bone mass in vivo, many studies have used peripheral quantitative CT (pQCT) [23,24]. The low resolutions, especially in slice thickness, used in this modality made it possible to perform in vivo imaging in small animals. Although the resolution inside a cross-section (in the order of 100 mm) is high enough to distinguish cortical and trabecular bone and to assess trabecular bone mass, the resolution is too low to resolve the trabecular architecture. The first in vivo high-resolution (<50 mm 3D) micro-CT images were made of rat tibiae using synchrotron radiation [2]. Advances have been made in the development of table-top micro-CT devices through the www.drugdiscoverytoday.com
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Figure 2. Simulating the bone renewal (remodeling) process in three dimensions using models created from micro-CT scans. The formation of cavities and refill of these cavities are simulated: resorption cavities are created with a random distribution over the surface of the trabeculae. Trabeculae that are disconnected are not repaired, all cavities that do not disconnect trabeculae are refilled. The remodeling parameters (number of resorption cavities, resorption depth and formation deficit) were based on histological data from the literature. From the model, it was concluded that most of the bone loss with aging is due to the formation deficit (the negative bone balance in each remodeling unit: the amount of bone made by osteoblasts is normally slightly smaller than the amount of bone resorbed by osteoclasts [69–95%]), followed by bone loss because of breached trabeculae that were not repaired (1–21%).
introduction of rotating source-detector systems, in which the animal is placed in a fixed bed, similar to that used for clinical CT systems. Moreover, as both computing memory and the resolution of charge-coupled device (CCD) cameras increased, the voxel size that could be achieved using microCT scanners decreased. Current high-end scanners use up to 10 Megapixel cameras with voxel sizes below 5 mm. These technological improvements have enabled the development of table-top in vivo micro-CT scanners with resolutions in the order of 10–50 mm and radiation dosages below 1 Gy per scan [8,25,26]. Follow-up studies using such devices showed halted bone growth and loss of trabecular bone in hind-limb suspended rats in contrast to trabecular reorganization in normally aging rats [25], and revealed the similarity in the bone loss dynamics of rats after ovariectomy and normally aging rats [26,27]. Another study showed the feasibility of using in vivo micro-CT to detect the increase in trabecular thickness caused by treatment with parathyroid hormone (PTH) and the bone-protective effect of various antiresorptive agents [28]. Also, bone resorption and adaptation in the rat fibula after osteotomy have been monitored successfully in vivo [29], as have the subchondral bone changes occurring in a rat model of osteoarthritis [30]. One of the biggest advantages of in vivo imaging, as concluded in most of the above-mentioned studies, is the 216
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increase in statistical power, because treatment effects can be measured independently of biological base-line variations. Thus, it is possible to detect smaller effects in an earlier stage than is possible with cross-sectional imaging modalities, such as conventional micro-CT and histology. Related to this, fewer animals are needed to perform an experiment, which can reduce both the cost and the animal inconvenience of each experiment.
In vivo bone architecture dynamics The possibilities of in vivo micro-CT are increased further when image-registration techniques are used [26,27,29]. Image registration comprises a set of image processing techniques that are generally used to match, for example, clinical CT images from longitudinal studies or to match images obtained from different imaging modalities like CT, magnetic resonance imaging (MRI) and positron emission tomography (PET) or single photon emission computed tomography (SPECT) [30–32]. In short, these techniques iteratively reposition one scanned set of images with respect to another set, until a certain mathematical criterion is optimized and the two sets ‘match’ optimally. When applied to longitudinal scans obtained by in vivo micro-CT, two datasets of a bone of an individual animal can be matched such that they can be positioned on top of each other. Thus, it becomes possible to
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visualize and quantify where new bone is formed, and where old bone was resorbed ([26], Figs 3 and 4). This contrasts with traditional dynamic histomorphometry, which can only be used to measure new bone formation, obviously ex vivo. These registered images have also been used to observe the result of bone remodeling dynamics on single trabeculae [27]. It could be shown in a rat model of postmenopausal bone loss not only that are trabeculae resorbed but that the remaining trabeculae increase in thickness by apposition of new bone, and that several connected trabeculae remodel into thick trabecular struts (Fig. 5). Further, such longitudinal 3D datasets could be used to locally quantify changes in thickness (Fig. 6). The latter possibilities indicate the potential of in vivo micro-CT for drug-related research. Because the current paradigm in bone research is bone quality, which includes among
Figure 3. 3D longitudinal slab from the proximal tibia of an ovariectomized rat. Owing to ovariectomy there will be a reduction in estrogen levels and a subsequent loss of bone. This bone loss is often shown in the proximal femur of ovariectomized rats as a model for human osteoporosis. The figure shows the locations of bone resorption (yellow) and formation of new bone (red) in the first 4 weeks after ovariectomy. Clearly, the overall effects of ovariectomy are cancellous bone loss (mid-metphyseal part), but there are certain locations with bone apposition (e.g. the medial endo-cortex). These detailed analyses can elucidate the precise effects of different drugs that are under development or help to better understand existing drugs for osteoporosis.
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Figure 4. Fluorescent labeling confirms local sites of bone formation as suggested by in vivo micro-CT. (a) An histological section with a green fluorescent label that is incorporated at locations of new bone formation and (b) an in vivo measurement showing the same locations of bone formation and showing bone resorption locations in red and bone apposition in yellow that matches the green label in the histological section [26].
others bone architecture, there is a need to know how drugs that are used to influence bone metabolism affect trabecular architecture. Take, for instance, the ongoing debate on the bone anabolic effect of PTH treatment. It is generally accepted that PTH treatment can lead to the apposition of new bone on existing surfaces, but the debate focuses on the question of whether new bone struts can be formed that connect previously unconnected trabeculae. Although such actions have never been shown to really exist, recent in vivo micro-CT findings did show that bone remodeling is very dynamic and can transform existing trabecular structures into completely different structures. There is a need for caution when using in vivo micro-CT because of the influence of ionizing X-ray radiation on bone metabolism. Published studies report radiation doses of between 0.2 and 1.0 Gy per scan. Although relatively low, such a dose is still about 10,000 times greater than that generated by a general chest X-ray. A single dose as low as 0.25 Gy negatively affects bone marrow in humans [33] and thus caution is justified. Radiation doses below 1 Gy have been shown to affect rodent metabolism [34,35], although no effects of such doses have been found in bones and the epiphyseal growth-plates [36,37]. These studies indicate that a single dose of around 0.5 Gy is not likely to affect bone metabolism measurably. However, longitudinal studies involve multiple scans on the same animal, which increase radiation damage dependent on the number of the scans and the amount of time between the scanning sessions. Multiple scans of aged female wistar rats did not result in detectable differences in trabecular architecture [27,38]. In mice, however, multiple scans did result in detectable, although minor, www.drugdiscoverytoday.com
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Figure 5. Registered cross-sections from the tibial metaphysis of a rat. The subsequent in vivo measurements show the dynamics of bone remodeling as the rat ages from 1- to 2-year-old. Notice how remaining trabeculae increase in thickness and the remodeling of several thin trabeculae into one big rod.
bone loss in radiated legs relative to nonradiated contralateral legs [38,39]. The radio-sensitivity of the bones of experimental animals appears to depend on species, strains, age and bone site. In general, active proliferating cells are most sensitive to radiation, thus extra care needs to be taken with animals that have an active growth-plate and high remodeling rates.
Figure 6. Visualisation of local trabecular thickness in a 3D sample of metaphyseal trabecular bone in an ovariectomized rat at two time points (0 and 4 weeks after ovariectomy), and a visualization of the changes in thickness presented in the 4 week scan. Bone that was not present at week 0 (thus newly formed bone) is visualized transparent. Bone that remains becomes thicker (on average). The blue parts become thinner and will probably be removed in the next stage of the bone loss process.
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Conclusions In summary, micro-CT analysis has proven its value for bone morphometry and the estimation of bone stiffness and strength when it is combined with finite element computer models. In addition, the 3D reconstructions from micro-CT enable computer simulations of bone turnover that contribute to the understanding of the mechanisms of the bone metabolic process and its relation with mechanical parameters. Recent introduction of in vivo application of microCT showed the dynamic activities of single trabeculae and could in principle lead to a breakthrough for fast and accurate evaluation of drugs that effect bone metabolism. In particular for studies involving osteoporosis and osteoarthritis such an assessment is extremely useful. Progress can still be made with respect to decreasing the radiation dose. In particular, improvements in (micro-)CT reconstruction algorithms can be made. Such solutions can potentially improve the quality of the reconstructed data set especially when the images have a poor signal to noise ratio and thus allow shorter scanning time, similar to approaches in SPECT applications [40]. At the same time, there seems to be some progress in the development of monochromatic Xray beams, which might dramatically improve future X-ray (micro-)CT applications as well [41]. These combined novel software and hardware approaches might lead to new breakthroughs in micro-CT imaging, in particular for in vivo applications. Further developments in postprocessing software that interacts with micro-CT will provide more accurate information from the 3D images and can lead to highthroughput devices for fast phenotyping of small animals, which will be of great value in contemporary bone disease and other drug discovery programs.
Acknowledgements J.C.v.d.L. was supported by STW (Veni-program, RPG 6294) and J.H.W. was supported by the Dutch Arthritis Foundation. Part of the work presented was supported by the EU (grant QLRT-1999-02024, MIAB).
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