Beneath the Minerals, A Layer of Round Lipid Particles Was Identified To Mediate Collagen Calcification in Compact Bone Formation

¹ Florida Space Research Institute
² University of Pennsylvania

^* To whom correspondence should be addressed. E-mail: shaohua{at}fsri.org.

Submitted on November 3, 2005
Revised on January 27, 2006
Accepted on 25 July 2006

	Abstract

Astronauts lose 1-2% of their bone minerals per month during space flights. A systematic search for a countermeasure relies on a good understanding of the mechanism of bone formation at the molecular level. How collagen fibers, the dominant matrix protein in bones, are mineralized remains mysterious. Atomic force microscopy was carried out, in combination with immunostaining and Western blotting, on bovine tibia to identify unrecognized building blocks involved in bone formation and for an elucidation of the process of collagen calcification in bone formation. Before demineralization, tiles of hydroxyapatite crystals were found stacked along bundles of collagen fibers. These tiles were homogeneous in size and shape with dimensions 0.69 x 0.77 x 0.2 micron³. Demineralization dissolved these tiles and revealed small spheres with an apparent diameter around 145nm. These spheres appeared to be lipid particles since organic solvents dissolved them. The parallel collagen bundles had widths mostly less than 2 microns. Composition analysis of compact bones indicated a high content of apolar lipids, including triglycerides and cholesterol esters. Apolar lipids are known to form lipid droplets or lipoproteins, and these spheres are unlikely to be matrix vesicles as reported for collagen calcification in epiphyseal cartilages. Results from this study suggest the layer of round lipid particles on collagen fibers mediates the mineral deposition onto the fibers. The homogeneous size of these lipid particles and the presence of apolipoprotein in demineralized bone tissue suggest the possibility that these particles might be of lipoprotein origin. More studies are needed to verify the last claim and to exclude the possibility that they are secreted lipid droplets.

Key Words: atomic force microscopy, bone formation, bone lipidology, collagen calcification, osteoporosis, space medicine