Histochemical and Ultrastructural Studies on Relationship between Initial Calcification and Lipids

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This studies aimed at elucidating a biolgical relationship between ossification and lipids in the developmental process of the calvaria of newborn mice at electron microscopic level.Membranous bone tissues (parietal bone) of newborn mice were employed as the material in both in vivo (1-2 days of age) and in vitro (19 days of gestation) experimental systems. The results are as follows.(1) Even in the conventional double fixation method with glutaraldehyde and osmium tetroxide, a rapid dehydration procedure using cold acetone solutions after a perfusion fixation method (adjusted at a given pHin the fixative) made it possible to preserve at a higher frequency the substance which exhibits extracellular multilamellar structures.(2) On the basis of pH experiments for identification and the above findings, it is clear that the main constituents of osteoblastic lipid droplets are not phospholipids or triglycerides. Knowledge obtained strongly suggested that lipid droplets consisted of long-cha … More ined fatty acids.(3) As another component other than fatty acids contained in lipid droplets, the presence of mucopolysaccharides were also detected. There is also the possibility that thesubstance is a protein complex, but the bond with lipids was not thought tobe a strong bond as seen with the liver and intestine. The morphological relationship between above structures and so-called noncollagenous proteins such as osteocalcin orosteopontin etc. is now being studied.(4) With respect to extracellular lipids (in a droplet form) of osteoblasts, it is surmised to be composed of unionized (protonated) fatty acids, while ionized fatty acids-soap surrounds the lipid droplets. It should be emphasized that sudanophilic material (lipid-related sustance) described earlier as a substance observed at initial calcifying sites is likely to be unionized (protonated) fatty acids.(5) Since ionized fatty acids-soap has affinity for calcium, formation of fatty acid-calcium complexes were likely to occur.In conclusion, these results suggest that the presence of exracellular lipid droplets associated with osteoblasts in this study may play an important role in mineralization as a calcium-tapping mechanism, different from the matrix vesicle-associated calcification. Less