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Osteocytes  invivo  exhibit different functional states, but no specific marker to distinguish these is currently available. To simulate the differentiation process of pre-osteoblasts to osteocytes  invitro , MC3T3-E1 cells were cultured on type I collagen gel and a three-dimensional (3D) culture system was established. The Notch expression of osteocyte-like cells in 3D culture system was compared with that of  in situ  osteocytes in bone tissues. Immunohistochemistry demonstrated that Notch1 was not detected in “resting”  insitu  osteocytes, but was detected in normal cultured osteocyte-like cell line MLO-Y4. Osteocytes obtained from conventional osteogenic-induced osteoblasts and long-term cultured MLO-Y4 cells could not replicate the Notch1 expression pattern from  insitu  osteocytes. From day 14–35 of osteogenic induction, osteoblasts in 3D culture system gradually migrated into the gel to form canaliculus-like structures similar to bone canaliculus. On day 35, stellate-shaped osteocyte-like cells were observed, and expression of DMP1 and SOST, but not Runx2, was detected. Notch1 was not detected by immunohistochemistry, and  Notch1  mRNA level was not significantly different from that of  insitu  osteocytes. In MC3T3-E1 cells, down-regulation of  Notch2  increased  Notch1 , Notch downstream genes ( β-catenin  and  Nfatc1 ), and  Dmp1 . In MLO-Y4 cells, Notch2 decreased after  Notch1  siRNA transfection. Downregulation of  Notch1  or  Notch2  decreased  Nfatc1 ,  β-catenin , and  Dmp1 , and increased  Sost . We established “resting state” osteocytes using an  invitro  3D model. Notch1 can be a useful marker to help differentiate the functional states of osteocytes (activated vs. resting state).

Notch1 is a marker for in situ resting osteocytes in a 3-dimensional gel culture model