Establishment of an Osteoid Preosteocyte-like Cell MLO-A5 That Spontaneously Mineralizes in Culture

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Abstract
The mechanisms controlling the initiation of mineralization of bone matrix are not clear. To examine this process, we established a cell line called MLO‐A5 that mineralizes in sheets, not nodules, within 3 days of culture in the presence of β‐glycerophosphate (β‐GP) and ascorbic acid and within 7 days in the absence of β‐GP and ascorbic acid. The mineral formed in both cases was shown to be bonelike apatite by Fourier transformed infrared (FTIR) spectroscopy. Mineral‐to‐matrix ratios (min/matrix) calculated from the FTIR data, which are related directly to ash weight, were approximately 0.4 in the absence of β‐GP and ascorbic acid and approximately 1.2 in the presence of β‐GP and ascorbic acid. By comparison, these ratios in fetal rat calvarial cells without β‐GP equal 0 and with β‐GP 1.9. This cell line and three others (MLO‐A2, −D1, and −D6) were isolated from the long bones of transgenic mice expressing the large T‐antigen driven by the osteocalcin promoter, the same mice from which the osteocyte‐like cell line MLO‐Y4 was isolated. 1 The cell lines were selected based on a dendritic or stellate morphology. MLO‐A5 cells express high alkaline phosphatase, collagen type 1, parathyroid hormone/parathyroid hormone‐related peptide (PTH/PTHrP) receptor, bone sialoprotein (BSP), and osteocalcin (767 ng/106 cells compared with 6 cell for primary mouse osteoblasts and five osteoblast cell lines). The single unique feature of the MLO‐A5 cells compared with the other three nonmineralizing cell lines is the high expression of messenger RNA (mRNA) for BSP. These cell lines may represent stages of osteocyte differentiation and the MLO‐A5 cells represent the postosteoblast, preosteocyte responsible for triggering mineralization of osteoid.