BONE IS A LIVING tissue perpetually undergoing a metabolic process known as bone remodeling or bone turnover. The early developing stage of mineralized bone tissue in fetus (ossification) is a complex process in which an initial cartilage template is converted into bone.(1) Although such stages of skeleton development are, without a doubt, essential in determining the quality of the adult bone, several questions are still open about the processes related to the evolution and growth of bone tissue during ossification. Various techniques such as X-ray radiography,(2,3) histology,(4,5) and sonography,(6–8) have been used to probe the morphological development of bone during its formation. DXA(9,10) and quantitative computed tomography (QCT)(11) have been used to study the mineral density in developing vertebral bodies, showing an increase of bone mineral density (BMD) with fetal growth. Most of these techniques provide macroscopic information, but none of them are able to describe accurately the three-dimensional (3D) microscopic organization of trabecular components of bone or the mineral nature of calcified bone. Complementary techniques like X-ray diffraction (XRD),(12) electron or infrared microscopy,(13,14) and infrared spectroscopy(15) have been used to characterize the crystalline structure and the chemical composition of bone tissue. However, few studies have been performed on human samples,(16–18) and very rarely have they been performed on human vertebral fetal bone.(19)
The aim of this study was to give a deeper insight into the evolution process and microarchitectural and physical changes occurring in the early stages of skeleton formation in the human bone tissue. To this end we investigated the region of ossification centers in human fetal vertebra combining two techniques: X-ray microtomography and X-ray diffraction (XRD). These allowed the collection of morphological (micrometer scale) and crystallographic (nanometer and atomic scale) information.
Because of the scale of the bone structure, synchrotron radiation computed microtomography (SR-μCT) is extremely attractive for the 3D analysis of bone microarchitecture. Like conventional computed tomography, SR-μCT allows nondestructive 3D imaging of the internal structure of a sample.(20–22) The use of a third-generation X-ray synchrotron radiation source yields additional advantages: the high photon flux permits images up to very high spatial resolutions (<1 μm) and exceptional signal-to-noise ratios, while keeping reasonable acquisition times. Monochromatic X-ray beams are used, thus avoiding beam-hardening artifacts that are observed in images collected with conventional X-ray sources. As a consequence, accurate maps of the attenuation coefficient at the selected energy can be reconstructed. In addition, the possibility to finely tune the beam energy over a wide range permits the optimal choice of energy to improve the quality of images.(23)
The analysis of XRD patterns allows the description of the mineral structure of bone at an atomic and nanometer scale. Like microtomography, XRD also takes advantages of the high brilliance and the energy tunability of synchrotron radiation for collecting high-quality diffraction patterns. The use of a monochromatic X-ray beam gives simple line shape profile suitable for accurate line shape analysis and structural refinement. Moreover, the intense X-ray beam coupled to angle dispersed setups (bi-dimensional [2D] detector geometry) permits the collection of diffractograms with ample statistics in very limited time (from a few seconds to a few minutes).(24) Rapid data collection time is a substantial benefit compared with the time required with standard diffractometers based on single-detector scanning (several hours for each pattern). In addition, the reduction of collection time is advantageous for biological samples because it prevents an eventual degradation of the organic part during measurements. In this work, we used the advantages of SR XRD to collect high-quality diffractograms from bone samples to highlight the effect of age on the crystalline structure of mineralized bone.