Histochemical and Ultrastructural Studies on Relationship between Initial Calcification and Lipids

本研究旨在从电镜水平上阐明新生小鼠颅骨发育过程中骨化与脂质之间的生物学关系。在体内（1-2日龄）和体外（妊娠19日龄）实验系统中，采用新生小鼠的膜性骨组织（顶骨）作为材料。结果如下：(1)即使在传统的戊二醛和四氧化锇双重固定方法中，在灌注固定方法（在固定剂的给定pHin下调整）后使用冷丙酮溶液进行快速脱水程序，也可以在更高的频率下保存呈现细胞外多层结构的物质。(2)根据pH值鉴定实验和上述发现，可以明确成骨细胞脂滴的主要成分不是磷脂或甘油三酯。所获得的知识有力地表明，脂滴由长链、多链脂肪酸组成。(3)作为脂滴中除脂肪酸外的另一组分，还检测到粘多糖的存在。也有可能这种物质是一种蛋白质复合物，但与脂质的结合并不像与肝脏和肠道的结合那样紧密。目前正在研究上述结构与骨钙素、骨桥蛋白等所谓的非胶原蛋白之间的形态关系。(4)关于成骨细胞的细胞外脂质（以液滴形式），据推测它是由统一的（质子化的）脂肪酸组成的，而电离的脂肪酸——肥皂包围着脂滴。应该强调的是，之前描述的在初始钙化部位观察到的亲苏丹物质（脂类相关物质）很可能是统一（质子化）脂肪酸。(5)由于游离脂肪酸-肥皂对钙具有亲和力，因此很可能形成脂肪酸-钙络合物。总之，这些结果表明，本研究中与成骨细胞相关的细胞外脂滴的存在可能在矿化中发挥重要作用，作为一种钙攻壁机制，不同于基质囊泡相关的钙化。少

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