Skip to main content
SpringerLink
Log in

Isolated primary osteocytes express functional gap junctions in vitro

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

The osteocyte is the most abundant cell type in bone and is embedded in mineralized bone matrix. Osteocytes are still poorly characterized because of their location and the lack of primary osteocyte isolation methods. Data on the cell biology of osteocytes is especially limited. We have isolated primary osteocytes from rat cortical bone by applying repeated enzymatic digestion and decalcification. The isolated osteocytes expressed typical osteocytic morphology with cell-cell contacts via long protrusions after a 1-day culture. These cells were negative or faintly positive for alkaline phosphatase but expressed high levels of osteocalcin, PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome), and DMP1 (dentin matrix protein 1). These cells also revealed patchy membrane staining for connexin43. For studying the function of gap junctions in isolated osteocytes, we microinjected rhodamine-labeled dextran (MW: 10,000) and Lucifer yellow (MW: 457) and found that Lucifer yellow was rapidly transmitted to several surrounding cells, whereas dextran remained in the injected cells. Heptanol and 18α-glycyrrhetinic acid inhibited the transfer of Lucifer yellow. This clearly showed the existence of functional gap junctions in cultured osteocytes. Enveloped viruses, such as vesicular stomatitis virus and influenza A virus, were used for studying cell polarity. We were unable to demonstrate plasma membrane polarization with enveloped viruses in isolated primary osteocytes in culture. Our results suggest that osteocytes do not possess apical and basolateral plasma membrane domains as do osteoblasts, which are their precursors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Beck L, Soumounou Y, Martel J, Krishnamurthy G, Gauthier C, Goodyer CG, Tenenhouse HS (1997) Pex/PEX tissue distribution and evidence for a deletion in the 3′ region of the Pex gene in X-linked hypophosphatemic mice. J Clin Invest 99:1200–1209

    PubMed  CAS  Google Scholar 

  • Burger EH, Klein-Nulend J (1999) Mechanotransduction in bone-role of the lacuno-canalicular network. FASEB J 13 Suppl:S101–S112

    Google Scholar 

  • Cheng B, Kato Y, Zhao S, Luo J, Sprague E, Bonewald LF, Jiang JX (2001) PGE(2) is essential for gap junction-mediated intercellular communication between osteocyte-like MLO-Y4 cells in response to mechanical strain. Endocrinology 142:3464–3473

    Article  PubMed  CAS  Google Scholar 

  • Civitelli R, Beyer EC, Warlow PM, Robertson AJ, Geist ST, Steinberg TH (1993) Connexin43 mediates direct intercellular communication in human osteoblastic cell networks. J Clin Invest 91:1888–1896

    Article  PubMed  CAS  Google Scholar 

  • Davidson JS, Baumgarten IM, Harley EH (1986) Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid. Biochem Biophys Res Commun 134:29–36

    Article  PubMed  CAS  Google Scholar 

  • Donahue HJ (2000) Gap junctions and biophysical regulation of bone cell differentiation. Bone 26:417–422

    Article  PubMed  CAS  Google Scholar 

  • Doty SB (1981) Morphological evidence of gap junctions between bone cells. Calcif Tissue Int 33:509–512

    Article  PubMed  CAS  Google Scholar 

  • Du L, Desbarats M, Viel J, Glorieux FH, Cawthorn C, Ecarot B (1996) cDNA cloning of the murine Pex gene implicated in X-linked hypophosphatemia and evidence for expression in bone. Genomics 36:22–28

    Article  PubMed  CAS  Google Scholar 

  • George A, Sabsay B, Simonian PA, Veis A (1993) Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization. J Biol Chem 268:12624–12630

    PubMed  CAS  Google Scholar 

  • Guo R, Quarles LD (1997) Cloning and sequencing of human PEX from a bone cDNA library: evidence for its developmental stage-specific regulation in osteoblasts. J Bone Miner Res 12:1009–1017

    Article  PubMed  CAS  Google Scholar 

  • Hefley T, Cushing J, Brand JS (1981) Enzymatic isolation of cells from bone: cytotoxic enzymes of bacterial collagenase. Am J Physiol 240:C234–C238

    PubMed  CAS  Google Scholar 

  • Hefley TJ (1987) Utilization of FPLC-purified bacterial collagenase for the isolation of cells from bone. J Bone Miner Res 2:505–516

    PubMed  CAS  Google Scholar 

  • Horton AC, Ehlers MD (2003) Neuronal polarity and trafficking. Neuron 40:277–295

    Article  PubMed  CAS  Google Scholar 

  • Ilvesaro J, Metsikko K, Vaananen K, Tuukkanen J (1999) Polarity of osteoblasts and osteoblast-like UMR-108 cells. J Bone Miner Res 14:1338–1344

    Article  PubMed  CAS  Google Scholar 

  • Jande SS, Belanger LF (1973) The life cycle of the osteocyte. Clin Orthop 94:281–305

    PubMed  Google Scholar 

  • Kaplow LS (1955) A histochemical procedure for localizing and evaluating leukocyte alkaline phosphatase activity in smears of blood and marrow. Blood 10:1023–1029

    PubMed  CAS  Google Scholar 

  • Liu F, Malaval L, Aubin JE (1997) The mature osteoblast phenotype is characterized by extensive plasticity. Exp Cell Res 232:97–105

    Article  PubMed  CAS  Google Scholar 

  • Lombardi T, Montesano R, Orci L (1985) Polarized plasma membrane domains in cultured endothelial cells. Exp Cell Res 161:242–246

    Article  PubMed  CAS  Google Scholar 

  • Lombardi T, Montesano R, Wohlwend A, Amherdt M, Vassalli JD, Orci L (1985) Evidence for polarization of plasma membrane domains in pancreatic endocrine cells. Nature 313:694–696

    Article  PubMed  CAS  Google Scholar 

  • Marotti G, Farneti D, Remaggi F, Tartari F (1998) Morphometric investigation on osteocytes in human auditory ossicles. Anat Anz 180:449–453

    CAS  Google Scholar 

  • Martin K, Helenius A (1991) Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell 67:117–130

    Article  PubMed  CAS  Google Scholar 

  • McCarthy TL, Centrella M, Canalis E (1988) Further biochemical and molecular characterization of primary rat parietal bone cell cultures. J Bone Miner Res 3:401–408

    PubMed  CAS  Google Scholar 

  • Menton DN, Simmons DJ, Chang SL, Orr BY (1984) From bone lining cell to osteocyte-an SEM study. Anat Rec 209:29–39

    Article  PubMed  CAS  Google Scholar 

  • Metsikko K, Hentunen T, Vaananen K (1992) Local expression and exocytosis of viral glycoproteins in multinucleated muscle cells. J Cell Biol 117:987–995

    Article  PubMed  CAS  Google Scholar 

  • Mikuni-Takagaki Y, Kakai Y, Satoyoshi M, Kawano E, Suzuki Y, Kawase T, Saito S (1995) Matrix mineralization and the differentiation of osteocyte-like cells in culture. J Bone Miner Res 10:231–242

    PubMed  CAS  Google Scholar 

  • Nijweide PJ, Mulder RJ (1986) Identification of osteocytes in osteoblast-like cell cultures using a monoclonal antibody specifically directed against osteocytes. Histochemistry 84:342–347

    Article  PubMed  CAS  Google Scholar 

  • Palumbo C, Palazzini S, Marotti G (1990) Morphological study of intercellular junctions during osteocyte differentiation. Bone 11:401–406

    Article  PubMed  CAS  Google Scholar 

  • Palumbo C, Palazzini S, Zaffe D, Marotti G (1990) Osteocyte differentiation in the tibia of newborn rabbit: an ultrastructural study of the formation of cytoplasmic processes. Acta Anat (Basel) 137:350–358

    CAS  Google Scholar 

  • Palumbo C, Ferretti M, Marotti G (2004) Osteocyte dendrogenesis in static and dynamic bone formation: an ultrastructural study. Anat Rec 278A:474–480

    Article  Google Scholar 

  • Qin C, Brunn JC, Jones J, George A, Ramachandran A, Gorski JP, Butler WT (2001) A comparative study of sialic acid-rich proteins in rat bone and dentin. Eur J Oral Sci 109:133–141

    Article  PubMed  CAS  Google Scholar 

  • Rahkila P, Luukela V, Vaananen K, Metsikko K (1998) Differential targeting of vesicular stomatitis virus G protein and influenza virus hemagglutinin appears during myogenesis of L6 muscle cells. J Cell Biol 140:1101–1111

    Article  PubMed  CAS  Google Scholar 

  • Reilly TM, Seldes R, Luchetti W, Brighton CT (1998) Similarities in the phenotypic expression of pericytes and bone cells. Clin Orthop 346:95–103

    PubMed  Google Scholar 

  • Salo J, Lehenkari P, Mulari M, Metsikko K, Vaananen HK (1997) Removal of osteoclast bone resorption products by transcytosis. Science 276:270–273

    Article  PubMed  CAS  Google Scholar 

  • Schiller PC, Mehta PP, Roos BA, Howard GA (1992) Hormonal regulation of intercellular communication: parathyroid hormone increases connexin 43 gene expression and gap-junctional communication in osteoblastic cells. Mol Endocrinol 6:1433–1440

    Article  PubMed  CAS  Google Scholar 

  • Singh IJ, Tonna EA, Gandel CP (1974) A comparative histological study of mammalian bone. J Morphol 144:421–437

    Article  PubMed  CAS  Google Scholar 

  • Toyosawa S, Shintani S, Fujiwara T, Ooshima T, Sato A, Ijuhin N, Komori T (2001) Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts. J Bone Miner Res 16:2017–2026

    Article  PubMed  CAS  Google Scholar 

  • Plas A van der, Nijweide PJ (1992) Isolation and purification of osteocytes. J Bone Miner Res 7:389–396

    Article  PubMed  Google Scholar 

  • Plas A van der, Aarden EM, Feijen JH, Boer AH de, Wiltink A, Alblas MJ, Leij L de, Nijweide PJ (1994) Characteristics and properties of osteocytes in culture. J Bone Miner Res 9:1697–704

    PubMed  Google Scholar 

  • Westbroek I, De Rooij KE, Nijweide PJ (2002) Osteocyte-specific monoclonal antibody MAb OB7.3 is directed against Phex protein. J Bone Miner Res 17:845–853

    Article  PubMed  CAS  Google Scholar 

  • Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, Shpektor D, Jonas M, Kovacevich BR, Staehling-Hampton K, Appleby M, Brunkow ME, Latham JA (2003) Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 22:6267–6276

    Article  PubMed  CAS  Google Scholar 

  • Wong G, Cohn DV (1974) Separation of parathyroid hormone and calcitonin-sensitive cells from non-responsive bone cells. Nature 252:713–715

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Professor Pirkko Härkönen for critical reading and comments on the manuscript.

Author information

Authors and Affiliations

  1. Department of Anatomy, Institute of Biomedicine, University of Turku, Turku, 20520, Finland

    Guoliang Gu, Martin Nars, Teuvo A. Hentunen & H. Kalervo Väänänen

  2. Drug Discovery Graduate School, Turku, Finland

    Guoliang Gu

  3. Department of Anatomy, University of Oulu, Oulu, Finland

    Kalervo Metsikkö

Authors
  1. Guoliang Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martin Nars
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Teuvo A. Hentunen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kalervo Metsikkö
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. H. Kalervo Väänänen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H. Kalervo Väänänen.

Additional information

This study was supported by the Academy of Finland, the Sigrid Juselius Foundation, and the Drug Discovery Graduate School.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gu, G., Nars, M., Hentunen, T.A. et al. Isolated primary osteocytes express functional gap junctions in vitro. Cell Tissue Res 323, 263–271 (2006). https://doi.org/10.1007/s00441-005-0066-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-005-0066-3

Keywords

Search

Navigation