Abstract
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.
Highlights
Osteocytes are the major cell type in the bone and account for up to 95% of bone cells [1]
The third approach was to pre-differentiate pre-osteoblasts for 7 days on tissue culture polystyrene (TCPS), using dexamethasone, ascorbic acid 2 phosphate, and β-glycerophosphate, before embedding them into a collagen gel and further cultivating them for 14 days. For all these approaches the quality of osteocytes was checked by morphological evaluation and gene expression analysis
Further characteristic of osteocytes is the expression of E11, PHEX, matrix extracellular phosphoglycoprotein (MEPE), sclerostin, RANKL, and osteocalcin
Summary
Osteocytes are the major cell type in the bone and account for up to 95% of bone cells [1]. The poor accessibility of osteocytes and their difficult cultivation have only recently made it possible to develop a suitable isolation method [3] and in vitro environment [4] It has been supposed, that osteocytes orchestrate bone homeostasis by regulating bone-forming osteoblasts and bone-resorbing osteoclasts. Interactions between osteoblasts and osteocytes have already been demonstrated: mature osteocytes express sclerostin and Dkk, which have a negative effect on the Wnt/β-catenin pathway. This pathway plays an important role in the regulation of bone mass by osteoblasts and its inhibition suppresses the activity of osteoblasts [5,6,7]. Conclusion: Interaction of osteoblasts and osteocytes can be monitored in an in vitro model, comprising solely primary human cells
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