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In Vitro Co-Culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels

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...

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Autores principales: Skottke, Jasmin, Gelinsky, Michael, Bernhardt, Anne
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514924/
https://www.ncbi.nlm.nih.gov/pubmed/31018582
http://dx.doi.org/10.3390/ijms20081998
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author Skottke, Jasmin
Gelinsky, Michael
Bernhardt, Anne
author_facet Skottke, Jasmin
Gelinsky, Michael
Bernhardt, Anne
author_sort Skottke, Jasmin
collection PubMed
description 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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spelling pubmed-65149242019-05-30 In Vitro Co-Culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels Skottke, Jasmin Gelinsky, Michael Bernhardt, Anne Int J Mol Sci Article 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. MDPI 2019-04-23 /pmc/articles/PMC6514924/ /pubmed/31018582 http://dx.doi.org/10.3390/ijms20081998 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Skottke, Jasmin
Gelinsky, Michael
Bernhardt, Anne
In Vitro Co-Culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels
title In Vitro Co-Culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels
title_full In Vitro Co-Culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels
title_fullStr In Vitro Co-Culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels
title_full_unstemmed In Vitro Co-Culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels
title_short In Vitro Co-Culture Model of Primary Human Osteoblasts and Osteocytes in Collagen Gels
title_sort in vitro co-culture model of primary human osteoblasts and osteocytes in collagen gels
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514924/
https://www.ncbi.nlm.nih.gov/pubmed/31018582
http://dx.doi.org/10.3390/ijms20081998
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