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RSS FeedsIJMS, Vol. 20, Pages 1998: In Vitro Co-culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels (International Journal of Molecular Sciences)

 
 

23 april 2019 17:02:32

 
IJMS, Vol. 20, Pages 1998: In Vitro Co-culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels (International Journal of Molecular Sciences)
 


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.


 
164 viewsCategory: Biochemistry, Biophysics, Molecular Biology
 
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IJMS, Vol. 20, Pages 1996: Two Sides of the Coin: Ezrin/Radixin/Moesin and Merlin Control Membrane Structure and Contact Inhibition (International Journal of Molecular Sciences)
 
 
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