In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria.

Abstract

We investigated the capacity'of a clonal osteogenic cell line MC3T3-EI, established from newborn mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the confluent state, to differentiate into osteoblasts and mineralize in vitro. The cells in the growing state showed a fibroblastic morphology and grew to form multiple layers. On day 21, clusters of cells exhibiting typical osteoblastic morphology were found in osmiophilic nodular regions. Such nodules increased in number and size with incubation time and became easily identifiable with the naked eye by day 40-50. In the central part of well-developed nodules, osteocytes were embedded in heavily mineralized bone matrix. Osteoblasts were arranged at the periphery of the bone spicules and were surrounded by lysosome-rich cells and a fibroblastic cell layer. Numerous matrix vesicles were scattered around the osteoblasts and young osteocytes. Matrix vesicles and plasma membranes of osteoblasts, young osteocytes, and lysosome-rich cells showed strong reaction to cytochemical stainings for ALP activity and calcium ions. Minerals were initially localized in the matrix vesicles and then deposited on well-banded collagen fibrils. Deposited minerals consisted exclusively of calcium and phosphorus, and some of the crystals had matured into hydroxyapatite crystals. These results indicate that MC3T3-EI cells have the capacity to differentiate into osteoblasts and osteocytes and to form calcified bone tissue in vitro.Recently, we established eight cell lines from newborn mouse calvaria and selected a clone, designated MC3T3-EI, from one of these lines on the basis of its high alkaline phosphatase (ALP) activity in the resting state (l). Cells of this clonal line had very low ALP activity in the growing state, but enzyme activity increased several hundredfold after cultures reached a confluent state. The cells produced abundant fibrous intercellular substances. In addition, intercellular spaces in day-30 cultures stained positively with alizarin red S. These observations suggest that MC3T3-EI cells are an osteogenic cell line, having the capacity to differentiate into osteoblasts and deposit minerals in vitro.We undertook the studies described here to further characterize the process of differentiation of MC3T3-E1 cells and calcification in vitro. We found that calcified bone tissue was formed in MC3T3-E 1 cultures by a process closely resembling that observed in intramembranous osteogenesis in vivo. Mineral deposits were identified as hydroxyapatite by energy dispersive x-ray analysis and electron diffraction analysis. MATERIALS AND METHODSCell Culture: The MC3T3-EI cells were grown in alpha modification of Eagle's minimal essential medium (a-MEM) (Flow Laboratories, McLean, VA) supplemented with 10% newborn calf serum (Flow Lab., Stanmore, New South Wales, Australia) and 60 lag/ml of kanamycin sulfate (Meiji Seika, Tokyo, Japan). CelLs were maintained at 37°C in a fully-humidified atmosphere of 5% CO2 in air and subcultured every 3 ...

Effects of arachidonic acid and docosahexaenoic acid on differentiation and mineralization of MC3T3‐E1 osteoblast‐like cells

“…48 In our experimental conditions MC3T3-E1 control cells exhibited a high basal ALP activity after 14 days, thereby confirming the results of others. 47 Compared with control, AA inhibited ALP activity in these cells after 14 days. These results are confirmed in a report by Yoshida et al 49 AA is the substrate for PGE 2 synthesis 50 and has been implicated in osteoblastic differentiation.…”

“…21,22,32,40 MC3T3-E1 murine osteoblast-like cells used in this study are considered immediate osteoblast precursors as these cells constitutively express high levels of ALP and calcify in basal conditions. 30,47 It has been shown by Igarishi et al that the ALP activity of control MC3T3-E1 cells increases after seeding, reaches a maximum on day 15, and decreases thereafter up to day 21. 48 In our experimental conditions MC3T3-E1 control cells exhibited a high basal ALP activity after 14 days, thereby confirming the results of others.…”

“…Nontransformed MC3T3-E1 mouse calvaria fibroblasts (established from the calvaria of an embryo/fetus C57BL/ 6 mouse), described to differentiate to osteoblasts, 31 were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany. Cell cultures were maintained in a-MEM (with 10% heatinactivated FCS) at 378C in a humidified atmosphere of 95% air and 5% CO 2 .…”

Transcriptional regulation of osteopontin gene in vivo by PEBP2αA/CBFA1 and ETS1 in the skeletal tissues

“…The MC3T3/E1 osteoblast cell line was generously provided by Dr Kodama (Sudo et al, 1983) and maintained in alpha modi®ed of Eagle's minimal essential medium (a-MEM; ICN Biomedicals, Costa Mesa, CA) supplemented with 10% FCS. The 293T human embryonic kidney cell line was kindly provided by Dr D Baltimore (Rockefeller University, New York, NY) and maintained in DMEM supplemented with 10% FCS.…”

Possible involvement of protein kinase C in proliferation and differentiation of osteoblast‐like cells

“…However, the involvement of protein kinase C in signal transduction system remains unclear. Therefore, we studied the effects of TPA and OAG, potent activators of protein kinase C [1], on DNA synthesis and ALP activity in osteoblast-like MC3T3-E1 cells cloned from newborn mouse calvaria [5,6]. We also studied the interaction of these protein kinase C activators with other osteoblast modulators, IGF-I [7,8] and PTH [9].…”