Age- and genotype-dependence of bone material properties in the osteogenesis imperfecta murine model (oim).

Cortical mineralization of long bones was studied in collagen alpha2(I)-deficient mice (oim) used as a model for human osteogenesis imperfecta. Aspects of the age development of the mice were characterized by combining nanometer- to micrometer-scale structural analysis with microhardness measurements. Bone structure was determined from homozygous (oim/oim) and heterozygous (oim/+) mice and their normal (+/+) littermates as a function of animal age by small-angle X-ray scattering (SAXS) and quantitative backscattered electron imaging (qBEI) measurements. SAXS studies found anomalies in the size and arrangement of bone mineral crystals in both homozygous and heterozygous mice aged 1-14 months. Generally, the crystals were smaller in thickness and less well aligned in these mice compared with control animals. An increase in the mean crystal thickness of the bone was found within all three genotypes up to an age of 3 months. Vicker's hardness measurements were significantly enhanced for oim bone (homozygotes and heterozygotes) compared with controls. The microhardness values were correlated directly with increased mineral content of homozygous and heterozygous compared with control bone, as determined by qBEI analysis. There was also a significant increase of mineral content with age. Two possibilities for collagen-mineral association are discussed for explaining the increased hardness and mineral content of oim/oim bone, together with its decreased toughness and thinner mineral crystals. As a consequence of the present measurements, one model for oim bone could incorporate small and densely packed mineral crystals. A second model for possible collagen-mineral association in oim material would consist of two families of mineral crystals, one being smaller and the other being much larger than the crystals found in normal mouse long bones.

Alendronate increases degree and uniformity of mineralization in cancellous bone and decreases the porosity in cortical bone of osteoporotic women.

The strength of bone is correlated with bone mass but is also influenced significantly by other factors such as structural properties of the matrix (e.g., collagen mutations) and the mineral. Changes at all levels of this organization could contribute to fracture risk. We investigated the effects of alendronate (Aln) treatment on the density of mineralization and the ultrastructure of the mineral/collagen composite, size and habitus of mineral particles in iliac cancellous bone, as well as on the porosity of iliac cortical bone from postmenopausal osteoporotic women. Twenty-four transiliac bone biopsies from Phase III Aln (10 mg/day) trials (placebo and Aln after 2 and 3 years of treatment, n = 6 per group) were studied. The mineral structure was investigated by quantitative backscattered electron imaging (qBEI) and by scanning small-angle X-ray scattering (scanning-SAXS). qBEI histograms reflect the bone mineralization density distribution (BMDD), whereas SAXS patterns characterize the size and arrangement of the mineral particles in bone. We found that: (i) the relative calcium content of osteoporotic bone was significantly lower than that of data-base controls; (ii) mineralization was significantly higher and more uniform after Aln treatment; (iii) size and habitus of the mineral particles was not different between placebo and Aln-treated groups; and (iv) the porosity of cortical bone was reduced significantly by Aln treatment. We conclude that Aln treatment increases the degree and uniformity of bone matrix mineralization without affecting the size and habitus of the mineral crystals. It also decreases the porosity of the corticalis. Together these effects may contribute to the observed reduction in fractures.

Structural development of the mineralized tissue in the human L4 vertebral body.

Knowledge of the structural development of the human vertebrae from non-weight-bearing before birth to weight-bearing after birth is still poor. We studied the mineralized tissue of the developing lumbar L4 vertebral body at ages 15 weeks postconception to 97 years from the tissue level (trabecular architecture) to the material level (micro- and nanostructure). Trabecular architecture was investigated by 2D histomorphometry and the material level was examined by quantitative backscattered electron imaging (for typical calcium content, CaMaxFreq) and scanning small-angle X-ray scattering (for mean mineral particle thickness). During early development, the trabecular orientation changed from a radial to a vertical/horizontal pattern. For bone area per tissue area and trabecular width in postnatal cancellous bone, the maximum was reached at adolescence (20 years), while for trabecular number the maximum was reached at childhood (approximately 1 year). CaMaxFreq was lower in early bone (approximately 21 wt%) than in mineralized cartilage (approximately 29 wt%) and adolescent bone (approximately 23 wt%). In conclusion, the changes at the tissue level were observed to continue throughout life while the development of bone at the material level (CaMaxFreq, mineral particle thickness and orientation) is essentially complete after the first years of life. CaMaxFreq and mean particle thickness increase rapidly during the first years and reach saturation. Remarkably, when these parameters are plotted versus logarithm of age, they appear linear.

Complementary information on bone ultrastructure from scanning small angle X-ray scattering and Fourier-transform infrared microspectroscopy.

Scanning small angle X-ray scattering (scanning SAXS) and Fourier-transform infrared microspectroscopy (FT-IRM) have previously been utilized independently to characterize the structural properties of bone in an anatomical position-resolved fashion. Whereas SAXS provides a direct measure of the physical characteristics of apatitic crystals, FT-IRM assesses structure of both mineral and organic matrix at the molecular level. In the present study both methods were applied to examine the same developing bone tissue from the L-4 vertebra of a 14-month-old (accidental death). A 200-microm-thick section was processed for examination by scanning electron microscopy and SAXS. Spectra were collected at 200 microm spatial resolution at specific locations in cortical and cancellous bone. Parameters determined included total SAXS intensity, crystal thickness (T), and degree and direction of predominant crystal orientation. For FT-IRM analysis, a section 4 microm thick was cut longitudinally from the top of the sample. Spectra of regions 100 x 100 microm2 were acquired from the same locations as the SAXS spectra. Integrated areas of the phosphate nu(1,3) collagen amide I, and carbonate nu2 absorbances, were calculated to obtain mineral: matrix and carbonate:mineral ratios. The relative quantities of types A, B, and labile carbonate (substituted for apatite hydroxyl, phosphate, and surface positions, respectively) were also evaluated. Polarized FT-IRM data were collected to determine molecular orientation of the apatite and collagen components. The results of this study show that the information obtained from the two techniques is complementary. Both SAXS and FT-IRM data revealed that the crystals were significantly larger in the cancellous region compared with the cortical region, that mineralization was greater in the cortex, and that the crystals were oriented to a larger degree in the cancellous compared with the cortical bone. The scanning SAXS measure of crystal thickness was significantly correlated to the FT-IRM measures of crystallinity, type A carbonate substitution, and crystal orientation. In conclusion, it was found that the combined use of SAXS and FT-IRM provides valuable, unique information on structural changes in bone at both the microstructural and ultrastructural level. Although each method can be used individually, the combination of techniques provides additional insights into the mechanism of bone crystal maturation.

Scanning small angle X-ray scattering analysis of human bone sections.

Scanning small-angle X-ray scattering (scanning SAXS) was applied for the first time on bone to compare results from SAXS directly with those from other position-sensitive methods, such as light and polarized light microscopy, back-scattered electron imaging, and radiographic imaging. Since scanning SAXS is a nondestructive method of investigation, images from all these techniques could be obtained from the same bone sections. Thus, it could be shown that both the collagen and the mineral crystals were predominantly aligned parallel to the trabeculae and, therefore, to principle stress directions. Moreover, the mean crystal thickness as determined by scanning SAXS was found to be different at various positions inside the trabecular and cortical structure. Finally, it could be shown that scanning SAXS is suitable for detecting local changes in bone material, e.g., due to fluoride treatment.