Abstract
Osteocytes are thought to be the mechanosensors of bone by sensing mechanical loads imposed upon the bone and transmitting these signals to the other bone cells to initiate bone modeling and remodeling. The location of osteocytes deep within bone is ideal for their function. However, this location makes the study of osteocytes in vivo technically difficult. There are several methods for obtaining and culturing primary osteocytes for in vitro experiments and ex vivo observation. In this chapter, several proven methods are discussed including the isolation of avian osteocytes from chicks and osteocytes from calvaria and long bones of young mice. A detailed protocol for the isolation of osteocytes from hypermineralized bone of mature and aged animals is provided.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bonewald LF (2011) The amazing osteocyte. J Bone Miner Res 26:229–238
Dallas SL, Prideaux M, Bonewald LF (2013) The osteocyte: an endocrine cell and more. Endocr Rev 34:658–690
van der Plas A, Nijweide PJ (1992) Isolation and purification of osteocytes. J Bone Miner Res 7:389–396
Tanaka K, Matsuo T, Ohta M et al (1995) Time-lapse microcinematography of osteocytes. Miner Electrolyte Metab 21:189–192
Aarden EM, Nijweide PJ, van der Plas A et al (1996) Adhesive properties of isolated chick osteocytes in vitro. Bone 18:305–313
Ajubi NE, Klein-Nulend J, Nijweide PJ et al (1996) Pulsating fluid flow increases prostaglandin production by cultured chicken osteocytes–a cytoskeleton-dependent process. Biochem Biophys Res Commun 225:62–68
Kamioka H, Honjo T, Takano-Yamamoto T (2001) A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy. Bone 28:145–149
Kamioka H, Ishihara Y, Ris H et al (2007) Primary cultures of chick osteocytes retain functional gap junctions between osteocytes and between osteocytes and osteoblasts. Microsc Microanal 13:108–117
Kamioka H, Sugawara Y, Murshid SA et al (2006) Fluid shear stress induces less calcium response in a single primary osteocyte than in a single osteoblast: implication of different focal adhesion formation. J Bone Miner Res 21:1012–1021
Klein-Nulend J, Semeins CM, Ajubi NE et al (1995) Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts–correlation with prostaglandin upregulation. Biochem Biophys Res Commun 217:640–648
Klein-Nulend J, van der Plas A, Semeins CM et al (1995) Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J 9:441–445
Westbroek I, Ajubi NE, Alblas MJ et al (2000) Differential stimulation of prostaglandin G/H synthase-2 in osteocytes and other osteogenic cells by pulsating fluid flow. Biochem Biophys Res Commun 268:414–419
Mikuni-Takagaki Y, Kakai Y, Satoyoshi M et al (1995) Matrix mineralization and the differentiation of osteocyte-like cells in culture. J Bone Miner Res 10:231–242
Kawata A, Mikuni-Takagaki Y (1998) Mechanotransduction in stretched osteocytes–temporal expression of immediate early and other genes. Biochem Biophys Res Commun 246:404–408
Mikuni-Takagaki Y (1999) Mechanical responses and signal transduction pathways in stretched osteocytes. J Bone Miner Metab 17:57–60
Mikuni-Takagaki Y, Suzuki Y, Kawase T et al (1996) Distinct responses of different populations of bone cells to mechanical stress. Endocrinology 137:2028–2035
Gu G, Hentunen TA, Nars M et al (2005) Estrogen protects primary osteocytes against glucocorticoid-induced apoptosis. Apoptosis 10:583–595
Kato Y, Boskey A, Spevak L et al (2001) Establishment of an osteoid preosteocyte-like cell MLO-A5 that spontaneously mineralizes in culture. J Bone Miner Res 16:1622–1633
Kato Y, Windle JJ, Koop BA et al (1997) Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res 12:2014–2023
Woo SM, Rosser J, Dusevich V et al (2011) Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo. J Bone Miner Res 26:2634–2646
Zhao S, Zhang YK, Harris S et al (2002) MLO-Y4 osteocyte-like cells support osteoclast formation and activation. J Bone Miner Res 17:2068–2079
Kramer I, Halleux C, Keller H et al (2010) Osteocyte Wnt/beta-catenin signaling is required for normal bone homeostasis. Mol Cell Biol 30:3071–3085
Nakashima T, Hayashi M, Fukunaga T et al (2011) Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat Med 17:1231–1234
Stern AR, Stern MM, Van Dyke ME et al (2012) Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice. BioTechniques 52:361–373
Jahn K, Lara-Castillo N, Brotto L et al (2012) Skeletal muscle secreted factors prevent glucocorticoid-induced osteocyte apoptosis through activation of beta-catenin. Eur Cell Mater 24:197–209
Kalajzic I, Matthews BG, Torreggiani E et al (2013) In vitro and in vivo approaches to study osteocyte biology. Bone 54:296–306
Qing H, Ardeshirpour L, Pajevic PD et al (2012) Demonstration of osteocytic perilacunar/canalicular remodeling in mice during lactation. J Bone Miner Res 27:1018–1029
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media. New York
About this protocol
Cite this protocol
Stern, A.R., Bonewald, L.F. (2015). Isolation of Osteocytes from Mature and Aged Murine Bone. In: Westendorf, J., van Wijnen, A. (eds) Osteoporosis and Osteoarthritis. Methods in Molecular Biology, vol 1226. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1619-1_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1619-1_1
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1618-4
Online ISBN: 978-1-4939-1619-1
eBook Packages: Springer Protocols