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Updated: Jan 25, 2026
Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts
Published on: May 13, 2014
In Vitro Co-culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels
Jasmin Skottke1, Michael Gelinsky2, Anne Bernhardt3
1Centre for Translational Bone, Joint and Soft Tissue Research, Medical Faculty and University Hospital, Technische Universität, 01307 Dresden, Germany. jasmin.skottke@tu-dresden.de.
Background:
Osteocytes are the key regulator cells in bone tissue, affecting activity of both osteoblasts and osteoclasts. Current in vitro studies on osteocyte-osteoblast interaction are invariably performed with rodent cells, mostly murine cell lines, which diminishes the clinical relevance of the data.
Objective:
The objective of the present study was to establish an in vitro co-culture system of osteoblasts and osteocytes, which is based solely on human primary cells.
Methods:
Three different approaches for the generation of human primary osteocytes were compared: direct isolation of osteocytes from bone tissue by multistep digestion, long-time differentiation of human pre-osteoblasts embedded in collagen gels, and short time differentiation of mature human osteoblasts in collagen gels. Co-cultivation of mature osteoblasts with osteocytes, derived from the three different approaches was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoblasts on the basal side of a porous membrane, which allowed the separate gene expression analysis for osteocytes and osteoblasts. Fluorescence microscopic imaging and gene expression analysis were performed separately for osteocytes and osteoblasts.
Results:
All examined approaches provided cells with typical osteocytic morphology, which expressed osteocyte markers E11, osteocalcin, phosphate regulating endopeptidase homolog, X-linked (PHEX), matrix extracellular phosphoglycoprotein (MEPE), sclerostin, and receptor activator of NF-κB Ligand (RANKL). Expression of osteocyte markers was not significantly changed in the presence of osteoblasts. In contrast, osteocalcin gene expression of osteoblasts was significantly upregulated in all examined co-cultures with differentiated osteocytes. Alkaline phosphatase (ALPL), bone sialoprotein II (BSPII), and RANKL expression of osteoblasts was not significantly changed in the co-culture.
Conclusion:
Interaction of osteoblasts and osteocytes can be monitored in an in vitro model, comprising solely primary human cells.
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