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

ABSTRACT

Purpose

Osteocytes in vivo 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 in vitro, 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.

Results

Immunohistochemistry demonstrated that Notch1 was not detected in “resting” in situ 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 in situ 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 in situ 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.

Conclusions

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

KEYWORDS:

• Osteocytes
• three-dimensional culture
• Notch1
• Notch2
• marker

Abbreviations

2D	=	two-dimenional
3D	=	three-dimensional
RANKL	=	nuclear factor-κB ligand
SOST	=	sclerostin
OPG	=	osteoprotegerin
BMSCs	=	bone marrow stromal cells
ALP	=	alkaline phosphatase
colla2	=	collagen type Ia2
PBS	=	phosphate-buffered saline
FBS	=	fetal bovine serum.

Author contributions

Hou-De Zhou designed the study; Ying-Hui Zhou, Jia-Yu Zhu, Yue Guo, Hao-Neng Tang, Fang Wang, Junaid Iqbal, Hui-Xuan Wu, Nan Hu, Fen Xiao, Ting Wang and Long Li collected data; Ying-Hui Zhou, Jia-Yu Zhu and Hou-De Zhou analyzed and interpreted the data. All authors participated in drafting of the article and approved the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.

Additional information

Funding

This work was supported by the National Natural Scientific Foundation of China [grant numbers: 82170900, 8217033609, 81770880, 81800788, 81970762], the Science & Technology Department of Hunan Province [grant numbers: 2020SK2080, 2015JC3012] and Changsha City [k1906019, kq1901118].