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Home Projects 1st call (2008) 6. High-throughput micro- and nano-tomographic phenotyping for characterization of bone ultrastructure and quality using synchrotron light

6. High-throughput micro- and nano-tomographic phenotyping for characterization of bone ultrastructure and quality using synchrotron light

Dr. M. Stampanoni (PSI/ETHZ), Dr. R. Müller (ETHZ), Dr. J.-P. Thiran (EPFL) - PhD student: Kevin Mader

Project finished in March 2013.

Thanks to recently developed genome-wide screening techniques major research efforts have been started to identify the genetic influence on components of the skeletal system. Animal models, especially the mouse, are critical for these studies, as the environmental influences can be largely controlled. This project deals with the development of novel high-throughput phenotyping procedures for the characterization of murine bone ultrastructure and bone quality using synchrotron radiation based micro- and nano-computed tomography (SR µCT and nCT). With the advent of third generation synchrotron radiation sources, tomography even in the submicrometer domain has become feasible and is employed to analyze trabecular architecture and local bone tissue properties down to the cellular level. The main biological goal of the project is to apply this novel morphometric analysis of the ultrastructural phenotypes as well as the study of the relationships between these phenotypes for the large number of samples, which are usually required for genetic studies. This will provide new insights in the assessment of bone quality on all levels of bone hierarchy but especially on the cellular level, where such an analysis will be performed for the first time ever in a linkage study.

Scientific background

Osteoporosis is primarily a disease of bone fragility resulting from decreased bone mass. In addition, altered architectural arrangement of bone tissue and impaired bone quality leads to decreased skeletal strength and increased fracture risk [1]. Bone mineral density (BMD), a measure of bone mass, has been identified in several epidemiological studies as being the single most important risk factor for osteoporotic fractures [2]. Recent data have shown that predicting trabecular bone strength can be greatly improved by including microarchitectural parameters in the analysis [3]. Further improvement in evaluating bone strength can be achieved by considering the cortical bone compartment for the prediction of bone strength. While the focal point of hip fracture studies was on trabecular bone for the last decades, cortical bone contributes significantly to the mechanical strength of bone [4]. Moreover, regarding the stiffness of cortical bone, the influence of small changes in the amount or density of bone tissue is even more pronounced than similar changes would exert in trabecular bone [5]. Eventually, loss of cortical rather than loss of trabecular bone predominates in cases of proximal femur fracture, which is among the most devastating of all osteoporotic fractures. On this account, cortical bone tissue has been investigated in more detail and cortical bone strength has been related primarily to BMD and other parameters, which describe cortical geometry [6]. However, BMD was reported to be related only weakly to the mechanical properties of cortical bone [7]. On the other hand, cortical geometry and particularly intracortical porosity has been shown already before to be linked to stiffness and strength of cortical bone specimens from human donors and from different vertebrates. Furthermore, intracortical porosity has been associated with fracture risk of patients with femoral neck fractures [8]. Nevertheless, measurement of cortical bone properties has been hampered by missing imaging tools to assess ultrastructural properties of the cortex including vascular and cellular void spaces.


Project outline

This project will investigate cortical bone morphology in more detail as an important aspect of bone quality. To this end a hierarchical approach will be followed to assess cortical bone tissue properties in different regimes of spatial resolution, beginning at the organ level and going down to the cellular domain (Figure 1).

Figure 1: Three different steps of the hierarchical imaging approach. Left: the whole mouse femur, scanned at low resolution (20 μm) on a tabletop device. Center: High-resolution (3.5 μm) image obtained at the synchrotron showing the vascular network in the cortical bone Right: ultra-high resolution (350 nm) showing small details of the vascular network and the distribution of the osteocyte lacunae. The scale bar is 1 mm, 200 microns and 50 microns respectively.

Thanks to recently developed genome-wide screening techniques major research efforts have been started to identify the genetic influence on components of the skeletal system. Animal models are critical for these studies, as the environmental influences can be largely controlled. Because of the recent deciphering of the mouse genome and the high homology between mouse and human genome, the mouse is a perfect model to study the influence of different genes on skeletal phenotypes. Inbred strains are particularly useful, because the genetic make-up is identical for each animal, yet, there is genetic difference between strains. This can be exploited using genetic breeding strategies for two genetically distinct strains. Since the number of animals per study can be very high, analytical methods need to be fully automated and user independent to efficiently assess bone structure and function. Despite the enormous data volume on the order of terabytes, approaches of high-throughput phenotypic characterization (phenomics) hold high promises as they provide enabling technology without any reliable link between genotype and phenotype can be made.

For this project we will focus on the porosity within cortical bone by using synchrotron radiation based micro- and nano-computed tomography (SR µCT and nCT). With the advent of third generation synchrotron radiation sources, tomography even in the submicrometer domain has become feasible and has been employed to analyze trabecular architecture and local bone tissue properties. Recent results show that the canal network, as a quantitative phenotype, is a major contributor to local tissue porosity, and therefore, can directly be linked to measures of bone tissue quality and with that, to the mechanical properties of bone. Additionally, osteocyte lacunae are believed to act as stress concentrators within tissue of compact bone.

In a preliminary study, a strategy to subdivide murine intracortical porosity into novel 3D ultrastructural phenotypes, namely the canal network and the osteocyte lacunar system, and to provide methods to volumetrically quantify these phenotypes for subsequent morphometric analysis has been established.

The main biological goal of the project is to apply this novel morphometric analysis of the ultrastructural phenotypes as well as the study of the relationships between these phenotypes for the large number of samples, which are usually required for genetic studies. We believe that this will provide new insights in the assessment of bone quality on all levels of bone hierarchy but especially on the cellular level, where such an analysis will be performed for the first time ever in a linkage study.

These novel and innovative methods will be applied to a large set of femurs from 2,000 mice for which the genetic make-up is known. Because bone quality is under genetic control, this approach allows determination of genetic regulatory loci that influence ultrastructural bone phenotypes. Such an imaging setup that is not available anywhere in the world is truly paradigm shifting since it allows for the first time to assess vascular and cellular bone properties at the micro- and nanoscopic level fully non-destructively with acceptable turn around times to allow genetic linkage studies with these large numbers of animals. To partition bone quality into its regulatory pathways, we propose to identify genes that regulate femur bone quality independent of growth hormone (GH) and insulin-like growth factor-1 (IGF-1), which are known to be of tremendous importance during bone growth. A spontaneous mutation in the mouse known as little is used to simplify the vertebral bone phenotype. In homozygous lit/lit mice, GH is not detectable, IGF-1 is fixed at low levels, and bone strength is reduced. In a new congenic little mouse we have shown [9] that femoral bone quality can vary independently of IGF-1 and that femoral bone quality is gender specific. By crossing these two strains, it will be possible to locate regulatory loci for adult bone quality in the absence of GH and in the presence of low IGF-1. We will also analyze bone structure-function relationships in detail and relate the findings to specific genetic locations using congenic and transgenic approaches. These ultrastructural data will be combined with data on genetic markers as obtained from genome-wide screening, allowing determination of chromosomal regions (quantitative trait loci, QTL) influencing bone quality.

References

[1] Anonymous 2001 Osteoporosis prevention, diagnosis, and therapy. Jama-Journal of the American Medical Association 285(6):785-795.

[2] Hui SL, Slemenda CW, Johnston CC 1988 Age and Bone Mass as Predictors of Fracture in a Prospective-Study. Journal of Clinical Investigation 81(6):1804-1809.

[3] Turner CH, Cowin SC, Rho JY, Ashman RB, Rice JC 1990 The fabric dependence of the orthotropic elastic constants of cancellous bone. Journal of Biomechanics 23(6):549-61.

[4] Mazess RB 1990 Fracture Risk - a Role for Compact-Bone. Calcified Tissue International 47(4):191-193.

5] Schaffler MB, Burr DB 1988 Stiffness of compact bone: effects of porosity and density. Journal of Biomechanics 21(1):13-6.

[6] Augat P, Reeb H, Claes LE 1996 Prediction of fracture load at different skeletal sites by geometric properties of the cortical shell. Journal of Bone Mineral Research 11(9):1356-63.[7] Snyder SM, Schneider E 1991 Estimation of mechanical properties of cortical bone by computed tomography. Journal of Orthopedic Research 9(3):422-31.

[8] Barth RW, Williams JL, Kaplan FS 1992 Osteon morphometry in females with femoral neck fractures. Clinical Orthopedics and Related Research (283):178-86.

[9] Schneider P. et al., “Ultrastructural Properties in Cortical Bone Vary Greatly in Two Inbred Strains of Mice as Assessed by Synchrotron Light Based Micro- and Nano-Computed Tomography” sub. to Journal of Bone Mineral Research.

Contact. Kevin Mader

ABSTRACT NCCBI MEETING 2010

ABSTRACT NCCBI MEETING 2011

 
Home Projects 1st call (2008) 6. High-throughput micro- and nano-tomographic phenotyping for characterization of bone ultrastructure and quality using synchrotron light