The proliferation and differentiation of osteoblasts in co-culture with human umbilical vein endothelial cells: An improved analysis using fluorescence-activated cell sorting |
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Authors: | Yu Zhang Andreas Schedle Michael Matejka Xiaohui Rausch-Fan Oleh Andrukhov |
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Institution: | (1) Department of Pediatrics, University of Virginia, Charlottesville, VA 22908, USA;(2) Department of Pediatrics, University of Ulm, 89075 Ulm, Germany;(3) Unité 246, Faculty of Medicine Xavier Bichat , INSERM, BP 416, 75870 Paris, Cedex 18, France;(4) Department of Pediatrics, University of Virginia Health Sciences Center, PO Box 800386, Charlottesville, VA 22908, USA; |
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Abstract: | The interaction of osteoblasts and endothelial cells plays a pivotal role in osteogenesis. This interaction has been extensively
studied using their direct co-culture in vitro. However, co-culture experiments require clear discrimination between the two different cell types in the mixture, but this
was rarely achieved. This study is the first to use fluorescence-activated cell sorting (FACS) for the separation and quantitative
analysis of the proliferation and differentiation of MG-63 cells grown in direct co-culture with human umbilical vein endothelial
cells (HUVECs). The cells of the MG-63 cell line have properties consistent with the characteristics of normal osteoblasts.
We labeled HUVECs with fluorescent antibody against CD31 and used FACS to measure the proportions of each cell type and to
separate them based on their different fluorescence intensities. The rate of proliferation of the MG-63 cells was estimated
based on a count of the total viable cells and the proportion of MG-63 cells in the mixture. The mRNA expression levels of
the osteoblast differentiation markers alkaline phosphatase (ALP), collagen type 1 (Coll-1) and osteocalcin (OC) in the MG-63
cells were measured via real-time PCR after the separation via FACS. We found that HUVECs stimulated the proliferation of
the MG-63 cells after 72 h of co-culture, and inhibited it after 120 h of co-culture. The mRNA expression levels of ALP and
Coll-1 significantly increased, whereas that of OC significantly decreased in MG-63 after co-culture with HUVECs. Using FACS
for the quantitative analysis of the proliferation and differentiation of osteoblasts directly interacting with endothelial
cells could have merit for further co-culture research. |
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