Cargando…
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...
Autores principales: | , , |
---|---|
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 |
_version_ | 1783417973239709696 |
---|---|
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. |
format | Online Article Text |
id | pubmed-6514924 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT skottkejasmin invitrococulturemodelofprimaryhumanosteoblastsandosteocytesincollagengels AT gelinskymichael invitrococulturemodelofprimaryhumanosteoblastsandosteocytesincollagengels AT bernhardtanne invitrococulturemodelofprimaryhumanosteoblastsandosteocytesincollagengels |