Calcified Tissue International

, Volume 75, Issue 3, pp 253–261 | Cite as

Osteoblast-like Cells Complete Osteoclastic Bone Resorption and Form New Mineralized Bone Matrix In Vitro

  • M. T. K. Mulari
  • Q. Qu
  • P. L. Härkönen
  • H. K. Väänänen
Article

Abstract

Bone remodeling involves old bone resorption by osteoclasts and new bone formation by osteoblasts. However, the precise cellular mechanisms underlying these consecutive events remain obscure. To address this question in vitro, we have established a cell culture model in which the resorption lacunae are first created by osteoclasts and osteoblast-like cells accomplish the subsequent bone formation. We isolated osteoclasts from rat bone marrow and cultured them on bovine bone slices for 48 hours to create resorption lacunae. After removing osteoclasts, confluent differentiated primary osteoblast cultures were trypsinized and the cells were replaced on the resorbed bone slices for up to 14 days. The cultures were then examined by confocal microscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM). Our data suggest that after osteoclastic bone resorption, osteoblast-like cells, not macrophages, remove the remaining organic matrix in the lacuna. After cleaning the lacuna, osteoblast-like cells deposit new collagen fibrils at the bottom of the lacuna and calcify the newly formed matrix only, as visualized by labeled tetracycline accumulation merely in the lacuna during the osteoblast culture. Furthermore, an electron-dense layer rich in osteopontin separates the old and new matrices suggesting formation of the cement line. Since the morphology of the newly formed matrix is similar to the natural bone with respect to the cement line and osteoid formation as well as matrix mineralization, the present method provides for the first time a powerful in vitro method to study the cellular mechanisms leading to bone remodeling also in vivo.

Keywords

Bone remodeling Bone resorption Osteoblast Osteoclast Osteopontin 

References

  1. 1.
    Eriksen, EF 1986Normal and pathological remodeling of human trabecular bone: three dimensional reconstruction of the remodeling sequence in normals and in metabolic bone disease.Endocr Rev7379408Google Scholar
  2. 2.
    Eriksen, EF, Gundersen, HJ, Melsen, F, Mosekilde, L 1984Reconstruction of the formative site in iliac trabecular bone in 20 normal individuals employing a kinetic model for matrix and mineral apposition.Metab Bone Dis Relat Res5243252Google Scholar
  3. 3.
    Frost, HM 1965A synchronous group of mammalian cells, whose in vivo behavior can be studied.Henry Ford Hosp Med Bull13161172Google Scholar
  4. 4.
    Jaworski, ZFG, Duck, B, Sekaly, G 1981Kinetics of osteoclasts and their nuclei in evolving secondary baversian systems.J Anat133397405Google Scholar
  5. 5.
    Kimmel, DB 1980A light microscopic description of osteoprogenitor cells of remodeling bone in the adult.Metab Bone Dis Rel Res2181Google Scholar
  6. 6.
    McKee, MD, Nanci, A 1996Osteopontin: an interfacial extracellular matrix protein in mineralized tissues.Connect Tissue Res35197205Google Scholar
  7. 7.
    Väänänen, HK, Zhao, H, Mulari, M, Halleen, JM 2000The cell biology of osteoclast function.J Cell Sci113377381Google Scholar
  8. 8.
    Yasuda, H, Shima, N, Nakagawa, N, Yamaguchi, K, Kinosaki, M, Goto, M, Mochizuki, SI, Tsuda, E, Morinaga, T, Udagawa, N, Takahashi, N, Suda, T, Higashio, K 1999A novel molecular mechanism modulating osteoclast differentiation and function.Bone25109113Google Scholar
  9. 9.
    Matsuzaki, K, Udagawa, N, Takahashi, N, Yamaguchi, K, Yasuda, H, Shima, N, Morinaga, T, Toyama, Y, Yabe, Y, Higashio, K, Suda, T 1998Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures.Biochem Biophys Res Commun246199204Google Scholar
  10. 10.
    Suda, T, Nakamura, I, Jimi, E, Takahashi, N 1997Regulation of osteoclast function.J Bone Miner Res12869879Google Scholar
  11. 11.
    Mundy, CR, Altman, AJ, Gondek, MD, Bandelin, JG 1977Direct resorption of bone by human monocytes.Science19611091111Google Scholar
  12. 12.
    Kahn, AJ, Stewart, CC, Teitelbaum, SL 1978Contact-mediated bone resorption by human monocytes in vitro.Science199988990Google Scholar
  13. 13.
    Quinn, JM, Athanasou, NA 1992Tumour infiltrating macrophages are capable of bone resorption.J Cell Sci101681686Google Scholar
  14. 14.
    Tran Van, PT, Vignery, A, Baron, R 1982Cellular kinetics of the bone remodeling sequence in the rat.Anat Rec202445451Google Scholar
  15. 15.
    Tran Van, P, Vignery, A, Baron, R 1982An electron-microscopic study of the bone-remodeling sequence in the rat.Cell Tissue Res225283292Google Scholar
  16. 16.
    Heersche, JN 1978Mechanism of osteoclastic bone resorption: a new hypothesis.Calcif Tissue Res268184Google Scholar
  17. 17.
    Rifkin, BR, Heijl, L 1979The occurrence of mononuclear cells at sites of osteoclastic bone resorption in experimental periodontitis.J Periodontol50636640Google Scholar
  18. 18.
    Chambers, TJ, Fuller, K 1985Bone cells predispose bone surfaces to resorption by exposure of mineral to osteoclastic contact.J Cell Sci76155165Google Scholar
  19. 19.
    Kusano, K, Miyaura, C, Inada, M, Tamura, T, Ito, A, Nagase, H, Kamoi, K, Suda, T 1998Regulation of matrix metalloproteinases (MMP −2, −3, −9, and −13) by interleukin-1 and interleukin-6 in mouse calvaria: association of MMP induction with bone resorption.Endocrinology13913381345Google Scholar
  20. 20.
    Meikle, MC, Bord, S, Hembry, RM, Compston, J, Croucher, PI, Reynolds, JJ 1992Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines.J Cell Sci10310931099Google Scholar
  21. 21.
    Winchester, SK, Block, SR, Fiacco, GJ, Partridge, NC 1999Regulation of expression of collagenase-3 in normal, differentiating rat osteoblasts.J Cell Physiol181479488Google Scholar
  22. 22.
    Everts, V, Delaisse, JM, Korper, W, Jansen, DC, Tigchelaar–Gutter, W, Saftig, P, Beertsen, W 2002The bone lining cell: its role in cleaning Howship’s lacunae and initiating bone formation.J Bone Miner Res177790Google Scholar
  23. 23.
    Selander, K, Lehenkari, P, Vaananen, HK 1994The effects of bisphosphonates on the resorption cycle of isolated osteoclasts.Calcif Tissue Int55368375Google Scholar
  24. 24.
    Lakkakorpi, P, Tuukkanen, J, Hentunen, T, Jarvelin, K, Vaananen, K 1989Organization of osteoclast microfilaments during the attachment to bone surface in vitro.J Bone Miner Res4817825Google Scholar
  25. 25.
    Qu, Q, Perala–Heape, M, Kapanen, A, Dahllund, J, Salo, J, Vaananen, HK, Harkonen, P 1998Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture.Bone22201209Google Scholar
  26. 26.
    Boyde, A, Ali, NN, Jones, SJ 1984Resorption of dentine by isolated osteoclasts in vitro.Br Dent J156216220Google Scholar
  27. 27.
    Chambers, TJ, Revell, PA, Fuller, K, Athanasou, NA 1984Resorption of bone by isolated rabbit osteoclasts.J Cell Sci66383399Google Scholar
  28. 28.
    Nesbitt, SA, Horton, MA 1997Trafficking of matrix collagens through bone-resorbing osteoclasts.Science276266269Google Scholar
  29. 29.
    Salo, J, Lehenkari, P, Mulari, M, Metsikko, K, Vaananen, HK 1997Removal of osteoclast bone resorption products by transcytosis.Science276270273Google Scholar
  30. 30.
    Chen, J, McKee, MD, Nanci, A, Sodek, J 1994Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin.Histochem J266768Google Scholar
  31. 31.
    Raisanen, SR, Halleen, J, Parikka, V, Vaananen, HK 2001Tartrate-resistant acid phosphatase facilitates hydroxyl radical formation and colocalizes with phagocytosed Staphylococcus aureus in alveolar macrophages.Biochem Biophys Res Commun288142150Google Scholar
  32. 32.
    Jones, SJ, Gray, C, Boyde, A 1994Simulation of bone resorption-repair coupling in vitro.Anat Embryol (Berl)190339349Google Scholar
  33. 33.
    Kasugai, S, Todescan, R,Jr, Nagata, T, Yao, KL, Butler, WT, Sodek, J 1991Expression of bone matrix proteins associated with mineralized tissue formation by adult rat bone marrow cells in vitro: inductive effects of dexamethasone on the osteoblastic phenotype.J Cell Physiol147111120Google Scholar
  34. 34.
    McKee, MD, Nanci, A 1996Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair.Microsc Res Tech33141164Google Scholar
  35. 35.
    McKee, MD, Nanci, A 1995Osteopontin and the bone remodeling sequence. Colloidal-gold immunocytochemistry of an interfacial extracellular matrix protein.Ann N Y Acad Sci760177189Google Scholar
  36. 36.
    Rittling, SR, Matsumoto, HN, McKee, MD, Nanci, A, An, XR, Novick, KE, Kowalski, AJ, Noda, M, Denhardt, DT 1998Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro.J Bone Miner Res1311011111Google Scholar
  37. 37.
    Dodds, RA, Connor, JR, James, IE, Rykaczewski, EL, Appelbaum, E, Dul, E, Gowen, M 1995Human osteoclasts, not osteoblasts, deposit osteopontin onto resorption surfaces: an in vitro and ex vivo study of remodeling bone.J Bone Miner Res1016661680Google Scholar
  38. 38.
    Flores, ME, Norgard, M, Heinegard, D, Reinholt, FP, Andersson, G 1992RGD-directed attachment of isolated rat osteoclasts to osteopontin, bone sialoprotein, and fibronectin.Exp Cell Res201526530Google Scholar
  39. 39.
    Reinholt, FP, Hultenby, K, Oldberg, A, Heinegard, D 1990Osteopontin—a possible anchor of osteoclasts to bone.Proc Natl Acad Sci USA8744734475Google Scholar
  40. 40.
    Ek–Rylander, B, Flores, M, Wendel, M, Heinegard, D, Andersson, G 1994Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. Modulation of osteoclast adhesion in vitro.J Biol Chem2691485314856Google Scholar
  41. 41.
    Ross, FP, Chappel, J, Alvarez, JI, Sander, D, Butler, WT, Farach–Carson, MC, Mintz, KA, Robey, PG, Teitelbaum, SL, Cheresh, DA 1993Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha v beta 3 potentiate bone resorption.J Biol Chem26899019907Google Scholar
  42. 42.
    Lakkakorpi, PT, Horton, MA, Helfrich, MH, Karhukorpi, EK, Vaananen, HK 1991Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface.J Cell Biol11511791186Google Scholar
  43. 43.
    Lucas, PA, Syftestad, GT, Caplan, AI 1986Partial isolation and characterization of a chemotactic factor from adult bovine bone for mesenchymal cells.Bone7365371Google Scholar
  44. 44.
    Mundy, GR, Poser, JW 1983Chemotactic activity of the gamma-carboxyglutamic acid containing protein in bone.Calcif Tissue Int35164168Google Scholar
  45. 45.
    Mundy, GR, Rodan, SB, Majeska, RJ, DeMartino, S, Trimmier, C, Martin, TJ, Rodan, GA 1982Unidirectional migration of osteosarcoma cells with osteoblast characteristics in response to products of bone resorption.Calcif Tissue Int34542546Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • M. T. K. Mulari
    • 1
  • Q. Qu
    • 2
  • P. L. Härkönen
    • 1
  • H. K. Väänänen
    • 1
  1. 1.Department of Anatomy and MediCity Research Laboratory, Institute of BiomedicineUniversity of TurkuTurkuFinland
  2. 2.Department of Liver SurgeryGeneral Post and Telecommunication Hospital/8th Teaching Hospital of Peking UniversityBeijingChina

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