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

Purpose: Osteocytes exhibit different functional states, but no specific marker to distinguish these is currently available.

Materials And Methods: To simulate the differentiation process of pre-osteoblasts to osteocytes , 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 osteocytes in bone tissues.

Results: Immunohistochemistry demonstrated that Notch1 was not detected in "resting" 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 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 mRNA level was not significantly different from that of osteocytes. In MC3T3-E1 cells, down-regulation of increased , Notch downstream genes ( and ), and . In MLO-Y4 cells, Notch2 decreased after siRNA transfection. Downregulation of or decreased , , and , and increased .

Conclusions: We established "resting state" osteocytes using an 3D model. Notch1 can be a useful marker to help differentiate the functional states of osteocytes (activated vs. resting state).