Isolation of calcifiable vesicles from aortas of rabbits fed with high cholesterol diets.

Advanced arterial wall calcification in atherosclerosis imposes a serious rupturing effect on the aorta. However, the mechanism of dystrophic calcification linked to hyperlipidemia, that causes atherosclerosis remains unknown. Emerging morphological and biochemical evidence reveals that calcifiable vesicles may have a role in plaque calcification. To determine whether a high cholesterol diet can induce arterial calcification and produce or activate calcifiable vesicles in aortas, a rabbit model was used. After 2 months of daily high lipid feeding (supplemented with 2% cholesterol and 6% peanut oil), typical atherosclerotic lesions developed. However, the mineral, if present in aortas, was insufficient to be detected by Fourier transform-infrared spectroscopy (FT-IR) or alizarin red staining, indicative of a non-calcifying stage of atherosclerosis. Small segments of thoracic aortas were digested in a crude collagenase solution to release calcifiable vesicles. Vesicles were also isolated from normal aortas as control to consider the possibility that membrane vesicles may be produced by crude collagenase digestion, which could cause the degradation of some cells. Calcifiable vesicles were precipitated at 300,000 x g after subcellular particles were removed by centrifugation at 30,000 x g. Calcifiability of isolated vesicles was then tested using calcifying media containing physiological levels of Ca2+ and Pi and 1 mM ATP. Electron microscopic observations showed that the isolated vesicles were heterogeneous in size and shape and capable of depositing electron dense particles. Fourier transform infrared spectroscopic analysis of the deposited particles revealed the presence of an amorphous mineral phase. The spectroscopic mineral to matrix ratios, related to the amount of mineralization, indicated that vesicles from cholesterol-fed rabbits produced more minerals than control vesicles obtained from the normal aortas. Alizarin red staining for mineral further demonstrated substantially higher calcifiability of the experimental vesicles. A 3-5 h exposure of the vesicles to calcifying media caused significant deposition of 45Ca and 32Pi in a vesicle protein-concentration dependent manner. Similar to previously reported observations with human atherosclerotic aorta vesicles, rabbit vesicles were enriched in ATP-hydrolyzing enzymes including Mg2+- or Ca2+-ATPase and NTP pyrophosphohydrolase that are implicated in normal and pathological calcification. Altogether, these observations suggest that accumulation of the released calcifiable vesicles, as a result of high cholesterol diets, may have a role in dystrophic calcification in hyperlipidemia-related atherosclerosis.

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.

Potential role of bone morphogenetic proteins in fracture healing.

Since their discovery, bone morphogenetic proteins have held the promise for use in various orthopaedic diseases. One of the largest areas of likely application is the area of fracture repair. Although millions of fractures occur annually and the majority heal satisfactorily, 5% to 10% go on to delayed union or nonunion. Bone morphogenetic proteins may be able to improve bony healing in these conditions and perhaps enhance the healing of fractures that otherwise heal satisfactorily. This study examines the preclinical data to support the concept of enhancing bony healing and discusses the preliminary data from clinical trials using bone morphogenetic proteins to augment bony healing. Although the potential clinical uses of bone morphogenetic proteins in fracture healing remain significant, this potential has yet to be realized.

FT-IR microscopic mappings of early mineralization in chick limb bud mesenchymal cell cultures.

Chick limb bud mesenchymal cells differentiate into chondrocytes and form a cartilaginous matrix in culture. In this study, the mineral formed in different areas within cultures supplemented with 4 mM inorganic phosphate, or 2.5, 5.0, and 10 mM beta-glycerophosphate (beta GP), was characterized by Fourier-transform infrared (FT-IR) microscopy. The relative mineral-to-matrix ratios, and distribution of crystal sizes at specific locations throughout the matrix were measured from day 14 to day 30. The only mineral phase detected was a poorly crystalline apatite. Cultures receiving 4 mM inorganic phosphate had smaller crystals which were less randomly distributed around the cartilage nodules than those in the beta GP-treated cultures. beta GP-induced mineral consisted of larger, more perfect apatite crystals. In cultures receiving 5 or 10 mM beta GP, the relative mineral-to-matrix ratios (calculated from the integrated intensities of the phosphate and amide I bands, respectively) were higher than in the cultures with 4 mM inorganic phosphate or in the in vivo calcified chick cartilage.