Mechanism by which MLO-A5 Late Osteoblasts/Early Osteocytes Mineralize in Culture: Similarities with Mineralization of Lamellar Bone

Abstract

The mechanisms whereby bone mineralizes are unclear. To study this process, we used a cell line, MLO-A5, which has highly elevated expression of markers of the late osteoblast such as alkaline phosphatase, bone sialoprotein, parathyroid hormone type 1 receptor, and osteocalcin and will mineralize in sheets, not nodules. In culture, markers of osteocytes and dendricity increase with time, features of differentiation from a late osteoblast to an early osteocyte. Mineral formation was examined using transmission electron microscopy, scanning electron microscopy with energydispersive X-ray analysis, and atomic force microscopy. At 3-4 days of culture, spheres of approximately 20-50 nm containing calcium and phosphorus were observed budding from and associated with developing cellular projections. By 5-6 days, these calcified spheres were associated with collagen fibrils, where over time they continued to enlarge and to engulf the collagen network. Coalescence of these mineralized spheres and collagen-mediated mineralization were responsible for the mineralization of the matrix. Similar calcified spheres were observed in cultured fetal rat calvarial cells and in murine lamellar bone. We propose that osteoidosteocytes generate spherical structures that calcify during the budding process and are fully mineralized on their developing cellular processes. As the cellular process narrows in diameter, these mineralized structures become associated with and initiate collagen-mediated mineralization. KeywordsMLO-A5 cells; Mineralization; Osteoblast; Osteoid; Osteocyte Bone cells such as osteoblasts, osteoid-osteocytes, and osteocytes may play different roles in the initiation and regulation of bone mineralization. As early as 1976 and 1981, Bordier and coworkers [1] and Nijweide and coworkers [2] proposed that osteoid-osteocytes play an important role in the initiation and control of matrix calcification. Osteoid-osteocytes were described by Palumbo [3] in 1986 to be cells actively making matrix and calcifying this matrix. Like osteoblasts, their activity was polarized toward the mineralization front to which their cellular processes were oriented. Cellular processes oriented toward blood vessels only began to appear when mineralization began to spread around the cell. She described the cell body reducing in size in parallel with the formation of cytoplasmic processes. This reduction was about 30% at the osteoid-osteocyte stage and 70% with complete maturation of the osteocyte.Correspondence to: C. Barragan-Adjemian; E-mail: Barraganc@umkc.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptOwen [4] stated that during the time for an osteoblast to become an osteocyte, the cell has manufactured three times its own volume in matrix.Mikuni-Takagaki and colleagues [5] proposed that casein kinase II, produced in high amounts by osteoid-osteocytes and not osteoblasts, is responsible for phosphorylation of the matrix proteins necessary for mineralization. Phosphoproteins appear t...

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