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Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes

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Abstract

Cultures of isolated osteocytes may offer an appropriate system to study osteocyte function, since isolated osteocytes in culture behave very much like osteocytes in vivo. In this paper we studied the capacity of osteocytes to change their surrounding extracellular matrix by production of matrix proteins. With an immunocytochemical method we determined the presence of collagen type I, fibronectin, osteocalcin, osteopontin and osteonectin in cultures of isolated chicken osteocytes, osteoblasts and periosteal fibroblasts. In osteoblast and periosteal fibroblast cultures, large extracellular networks of collagen type I and fibronectin were formed, but in osteocyte populations, extracellular threads of collagen or fibronectin were only rarely found. The percentage of cells positive for osteocalcin, osteonectin and osteopontin in the Golgi apparatus, on the other hand, was highest in the osteocyte population. These results show that osteocytes have the ability to alter the composition of their surrounding extracellular matrix by producing matrix proteins. We suggest this property is of importance for the regulation of the calcification of the bone matrix immediately surrounding the cells. More importantly, as osteocytes depend for their role as mechanosensor cells on their interaction with matrix proteins, the adaptation of the surrounding matrix offers a way to regulate their response to mechanical loading.

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References

  • Aarden EM, Burger EH, Nijweide PJ (1994) Function of osteocytes in bone. J Cell Biochem 55:287–299

    Article  PubMed  CAS  Google Scholar 

  • Arai N, Ohya K, Ogura H (1993) Osteopontin mRNA expression during bone resorption: an in situ hybridization study of induced ectopic bone in the rat. Bone Miner 22:129–145

    PubMed  CAS  Google Scholar 

  • Bélanger LF (1969) Osteocytic osteolysis. Calcif Tissue Res 4:1–12

    Article  PubMed  Google Scholar 

  • Bianco P, Hayashi Y, Silvestrini G, Termine JD, Bonucci E (1985) Osteonectin and GLA-protein in calf bone: ultrastructural immunohistochemical localization using the protein A-gold method. Calcif Tissue Int 37:684–686

    PubMed  CAS  Google Scholar 

  • Bianco P, Silvestrini G, Termine JD, Bonucci E (1988) Immunohistochemical localization of osteonectin in developing human and calf bone using monoclonal antibodies. Calcif Tissue Int 43:155–161

    PubMed  CAS  Google Scholar 

  • Boivin G, Morel G, Lian JB, Anthoine-Terrier C, Dubois PM, Meunier PJ (1990) Localization of endogenous osteocalcin in neonatal rat bone and its absence in articular cartilage: effect of warfarin treatment. Virchows Archiv 417:505–512

    Article  PubMed  CAS  Google Scholar 

  • Bonucci E (1990) The ultrastructure of the osteocyte. In: Bonucci E, Motta PM (eds) Ultrastructure of skeletal tissues, Kluwer, Amsterdam, pp 223–237

    Google Scholar 

  • Boyde A (1980) Evidence against “osteocytic osteolysis”. Bone 2S:239–255

    Google Scholar 

  • Bronckers ALJJ, Gay S, Dimuzio MT, Butler WT (1985) Immunolocalization of gamma-carboxyglutamic acid containing proteins in developing rat bones. Collagen Rel Res 5:273–281

    CAS  Google Scholar 

  • Chen J, Zhang Q, McCulloch CAG, Sodek J (1991) Immunohistochemical localization of bone sialoprotein in fetal porcine bone tissues: comparisons with secreted phosphoprotein 1 (SPP-1, osteopontin) and SPARC (osteonectin). Histochem J 23:281–289

    Article  PubMed  CAS  Google Scholar 

  • Chen J, McCulloch CAG, Sodek J (1993a) Bone sialoprotein in developing porcine dental tissues: cellular expression and comparison of tissue localization with osteopontin and osteonectin. Arch Oral Biol, 38:241–249

    Article  PubMed  CAS  Google Scholar 

  • Chen J, Singh K, Mukherjee BB, Sodek J (1993b) Developmental expression of osteopontin (OPN) mRNA in rat tissues: evidence for a role for OPN in bone formation and resorption. Matrix 13:113–123

    PubMed  CAS  Google Scholar 

  • Chen J, McKee MD, Nanci A, Sodek J (1994) Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin. Biochem J 26:67–78

    Article  CAS  Google Scholar 

  • Chen Y, Bal BS, Gorski JP (1992) Calcium and collagen binding properties of osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 from bone. J Biol chem 267:24871–24878

    PubMed  CAS  Google Scholar 

  • Gerstenfeld LC, Gotoh Y, McKee MD, Nanci A, Landis WJ, Glimcher MJ (1990) Expression and ultrastructural immunolocalization of a major 66 kDa phosphoprotein synthesized by chicken osteoblasts during mineralization in vitro. Anat Rec 228:93–103

    Article  PubMed  CAS  Google Scholar 

  • Gotoh Y, Gerstenfeld LC, Glimcher MJ (1990), Identification and characterization of the major chicken bone phosphoprotein. Analysis of its synthesis by cultured embryonic chick osteoblasts. Eur J Biochem 187:49–58

    Article  PubMed  CAS  Google Scholar 

  • Groot CG, Danes JK, Blok J, Hoogendijk A, Hauschka PV (1986) Light and electron microscopic demonstration of osteocalcin antigenicity in embryonic and adult rat bone. Bone 7:379–385

    Article  PubMed  CAS  Google Scholar 

  • Grzesik WJ, Gehron Robey P (1994) Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro. J Bone Miner Res 9:487–496

    PubMed  CAS  Google Scholar 

  • Hauschka PV, Lian JB, Gallop PM (1975) Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue. Proc Natl Acad Sci USA 72:3925–3929

    Article  PubMed  CAS  Google Scholar 

  • Hauschka PV, Frenkel J, DeMuth R, Gundberg CM (1983) Presence of osteocalcin and related higher molecular weight 4-carboxyglutamic acid-containing proteins in developing bone. J Biol Chem 258:176–180

    PubMed  CAS  Google Scholar 

  • Hefley TJ (1987) Utilization of FPLC-purified bacterial collagenase for the isolation of cells from bone. J Bone Miner Res 2:502–516

    Google Scholar 

  • Hynes RO, Yamada KM (1982) Fibronectins: multifunctional modular glycoproteins. J Cell Biol 95:369–377

    Article  PubMed  CAS  Google Scholar 

  • Ikeda T, Nomura, S, Yamaguchi A, Suda T, Yoshiki S (1992) In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections. J Histochem Cytochem 40:1079–1088

    PubMed  CAS  Google Scholar 

  • Ingram RT, Clarke BL, Fisher LW, Fitzpatrick LA (1993) Distribution of noncollagenous proteins in the matrix of adult human bone: evidence of anatomic and functional heterogeneity. J Bone Miner Res 8:1019–1029

    PubMed  CAS  Google Scholar 

  • Kasai R, Bianco, P, Gehron Robey P, Kahn AJ (1994) Production and characterization of an antibody against the human bone GLA protein (BGP/osteocalcin) propeptide and its use in immunocytochemistry of bone cells. Bone Miner 25:167–182

    Article  PubMed  CAS  Google Scholar 

  • Klein Nulend J, Plas A van der, Semeins CM, Ajubi NE, Frangos JA, Nijweide PJ, Burger EH (1995) Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9:441–445

    PubMed  CAS  Google Scholar 

  • Lanyon LE (1993) Osteocytes, strain detection, bone modeling and remodeling. Calcif Tissue Int 53(S1):S102-S107

    Article  PubMed  Google Scholar 

  • Majmudar G, Bole D, Goldstein SA, Bonadio J (1991) Bone cell culture in a three-dimensional polymer bead stabilizes the differentiated phenotype and provides evidence that osteoblastic cells synthesize type III collagen and fibronectin. J Bone Miner Res 6:869–881

    PubMed  CAS  Google Scholar 

  • Mark MP, Prince CW, Oosawa T, Gay S, Bronckers ALJJ, Butler WT (1987) Immunohistochemical demonstration of a 44 kDa phosphoprotein in developing rat bones. J Histochem Cytochem 35:707–715

    PubMed  CAS  Google Scholar 

  • Mark MP, Butler WT, Prince CW, Finkelman RD, Ruch J-V (1988) Developmental expression of 44-kDa bone phosphoprotein (osteospontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat. Differentiation 37:123–136

    Article  PubMed  CAS  Google Scholar 

  • Marotti G, Cane V, Palazzini S, Palumbo C (1990) Structure-function relationships in the osteocyte. Ital J Miner Electrolyte Metab 4:93–106

    Google Scholar 

  • McKee MD, Glimcher MJ, Nanci A (1992) High-resolution immunolocalization of osteopontin and osteocalcin in bone and cartilage during endochondral ossification in the chicken tibia. Anat Rec 234:479–492

    Article  PubMed  CAS  Google Scholar 

  • McKee MD, Farach-Carson MC, Butler WT, Hauschka PV, Nanci A (1993) Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and α2HS-glycoprotein) proteins in rat bone. J Bone Miner Res 8:485–496

    PubMed  CAS  Google Scholar 

  • Metsäranta M, Young MF, Sandberg M, Termine J, Vuorio E (1989) Localization of osteonectin expression in human skeletal tissues by in situ hybridization. Calcif Tissue Int 45:146–152

    PubMed  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 RJP (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 

  • Oldberg Å, Franzén A, Heinegård D (1986) Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc Natl Acad Sci USA 83:8819–8823

    Article  PubMed  CAS  Google Scholar 

  • Pacifici M, Oshima O, Fisher LW, Young MF, Shapiro IM, Leboy PS (1990) Changes in osteonectin distribution and levels are associated with mineralization of the chicken tibial growth cartilage. Calcif Tissue Int 47:51–61

    PubMed  CAS  Google Scholar 

  • Parfitt AM (1977) The cellular basis of bone turnover and bone loss. Clin Orhtop 127:236–247

    Google Scholar 

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

    PubMed  Google Scholar 

  • Plas A van der, Aarden EM, Feijen JHM, Boer AH de, Wiltink A, Alblas MJ, Leij L de, Nijweide PJ (1994) Characteristics and properties of osteocytes in culture. J Bone Miner Res 9:1697–1704

    Article  PubMed  Google Scholar 

  • Price PA, Otsuka AS, Poser JW, Kristaponis J, Raman N (1976) Characterization of a gamma-carboxyglutamic acid-containing protein from bone. Proc Natl Acad Sci USA 73:1447–1451

    Article  PubMed  CAS  Google Scholar 

  • Roach HI (1992)Trans-differentiation of hypertrophic chondrocytes into cells capable of producing a mineralized bone matrix. Bone Miner 19:1–20

    Article  PubMed  CAS  Google Scholar 

  • Roach HI (1994) Why does bone matrix contain non-collagenous proteins? The possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone mineralisation and resorption. Cell Biol Int 18:617–628

    Article  PubMed  CAS  Google Scholar 

  • Rubin GT, Mcleod KJ, Bain SD (1990) Functional strains and cortical bone adaptation: epigenetic assurance of skeletal integrity. J Biomech 23:43–54

    Article  PubMed  Google Scholar 

  • Sodek J, Chen J, Kasugai S, Nagata T, Zhang Q, Shapiro HS, Wrana JL, Goldberg HA (1992) Sialoproteins in bone remodelling. In: Davidovitch Z (ed) The biological mechanisms of tooth movement and craniofacial adaptation. The Ohio State University, College of Dentistry, Columbus; pp 127–136

    Google Scholar 

  • Sun Y-Q, McLeod KJ, Rubin CT (1995) Mechanically induced periosteal bone formation is paralleled by the upregulation of collagen type one mRNA in osteocytes as measured by in situ reverse transcript-polymerase chain reaction. Calcif Tissue Int 57:456–462

    Article  PubMed  CAS  Google Scholar 

  • Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML, Martin GR (1981) Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26:99–105

    Article  PubMed  CAS  Google Scholar 

  • Vermeulen AHM, Vermeer C, Bosman FT (1989) Histochemical detection of osteocalcin in normal and pathological bone. J Histochem Cytochem 37:1503–1508

    PubMed  CAS  Google Scholar 

  • Weiss RE, Reddi AH (1981) Appearance of fibronectin during the differentiation of cartilage, bone, and bone marrow. J Cell Biol 88:630–636

    Article  PubMed  CAS  Google Scholar 

  • Yamada KM (1983) Cell surface interactions with extracellular materials. Annu Rev Biochem 52:761–799

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Laboratory of Cell Biology and Histology, Department of Cell Biology, Faculty of Medicine, Leiden University, PO Box 2156, NL-2301 CD, Leiden, The Netherlands

    E. M. Aarden, A. -M. M. Wassenaar, M. J. Alblas & P. J. Nijweide

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  1. E. M. Aarden
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  2. A. -M. M. Wassenaar
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  3. M. J. Alblas
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  4. P. J. Nijweide
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Aarden, E.M., Wassenaar, A.M.M., Alblas, M.J. et al. Immunocytochemical demonstration of extracellular matrix proteins in isolated osteocytes. Histochem Cell Biol 106, 495–501 (1996). https://doi.org/10.1007/BF02473312

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  • DOI: https://doi.org/10.1007/BF02473312

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