Osteocytes form a three-dimensional (3D) cellular network within the mineralized bone matrix. The cellular network has important roles in mechanosensation and mechanotransduction related to bone homeostasis. We visualized the embedded osteocyte network in chick calvariae and observed the flow-induced Ca2+ signaling in osteocytes using 3D time-lapse imaging. In response to the flow, intracellular Ca2+ ([Ca2+]i) significantly increased in developmentally mature osteocytes in comparison with young osteocytes in the bone matrix. To investigate the differences in response between young and developmentally mature osteocytes in detail, we evaluated the expression of osteocyte-related genes using the osteocyte-like cell line MLO-Y4, which was 3D-cultured within type I collagen gels. We found that the c-Fos, Cx43, Panx3, Col1a1, and OCN mRNA levels significantly increased on day 15 in comparison with day 7. These findings indicate that developmentally mature osteocytes are more responsive to mechanical stress than young osteocytes and have important functions in bone formation and remodeling.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Power J, Loveridge N, Rushton N, Parker M, Reeve J (2002) Osteocyte density in aging subjects is enhanced in bone adjacent to remodeling haversian systems. Bone (NY) 30:859–865
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 (NY) 28:145–149
Gu G, Kurata K, Chen Z, Väänänen KH (2007) Osteocyte: a cellular basis for mechanotransduction in bone. J Biomech Sci Eng 2:150–165
Hu M, Tian GW, Gibbons DE, Jiao J, Qin YX (2015) Dynamic fluid flow induced mechanobiological modulation of in situ osteocyte calcium oscillations. Arch Biochem Biophys 579:55–61
Bonewald LF, Johnson ML (2008) Osteocytes, mechanosensing and Wnt signaling. Bone (NY) 42:606–615
Klein-Nulend J, Bakker AD, Bacabac RG, Vatsa A, Weinbaum S (2013) Mechanosensation and transduction in osteocytes. Bone (NY) 54:182–190
Komori T (2013) Functions of the osteocyte network in the regulation of bone mass. Cell Tissue Res 352:191–198
Bonewald LF (2006) Mechanosensation and transduction in osteocytes. Bonekey Osteovision 3:7–15
Hughes-Fulford M (2004) Signal transduction and mechanical stress. Sci STKE 2004(249):RE12
Wadhwa S, Godwin SL, Peterson DR, Epstein MA, Raisz LG, Pilbeam CC (2002) Fluid flow induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular signal-regulated kinase signaling pathway. J Bone Miner Res 17:266–274
Wadhwa S, Choudhary S, Voznesensky M, Epstein M, Raisz L, Pilbeam C (2002) Fluid flow induces COX-2 expression in MC3T3-E1 osteoblasts via a PKA signaling pathway. Biochem Biophys Res Commun 297:46–51
You J, Reilly GC, Zhen X, Yellowley CE, Chen Q, Donahue HJ, Jacobs CR (2001) Osteopontin gene regulation by oscillatory fluid flow via intracellular calcium mobilization and activation of mitogen-activated protein kinase in MC3T3-E1 osteoblasts. J Biol Chem 276:13365–13371
Berridge MJ, Bootman MD, Lipp P (1998) Calcium—a life and death signal. Nature (Lond) 395:645–648
Kamioka H, Sugawara Y, Murshid SA, Ishihara Y, Honjo T, Takano-Yamamoto T (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
Lu XL, Huo B, Chiang V, Guo XE (2012) Osteocytic network is more responsive in calcium signaling than osteoblastic network under fluid flow. J Bone Miner Res 27:563–574
Hung CT, Pollack SR, Reilly TM, Brighton CT (1995) Real-time calcium response of cultured bone cells to fluid flow. Clin Orthop Relat Res 313:256–269
Ishihara Y, Sugawara Y, Kamioka H, Kawanabe N, Hayano S, Balam TA, Naruse K, Yamashiro T (2013) Ex vivo real-time observation of Ca2+ signaling in living bone in response to shear stress applied on the bone surface. Bone (NY) 53:204–215
Xu H, Gu S, Riquelme MA, Burra S, Callaway D, Cheng H, Guda T, Schmitz J, Fajardo RJ, Werner SL, Zhao H, Shang P, Johnson ML, Bonewald LF, Jiang JX (2015) Connexin 43 channels are essential for normal bone structure and osteocyte viability. J Bone Miner Res 30:436–448
Ilvesaro J, Väänänen K, Tuukkanen J (2000) Bone-resorbing osteoclasts contain gap-junctional connexin-43. J Bone Miner Res 15:919–926
Burra S, Nicolella DP, Francis WL, Freitas CJ, Mueschke NJ, Poole K, Jiang JX (2010) Dendritic processes of osteocytes are mechanotransducers that induce the opening of hemichannels. Proc Natl Acad Sci USA 107:13648–13653
Cherian PP, Siller-Jackson AJ, Gu S, Wang X, Bonewald LF, Sprague E, Jiang JX (2005) Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin. Mol Biol Cell 16:3100–3106
Iwamoto T, Nakamura T, Doyle A, Ishikawa M, de Vega S, Fukumoto S, Yamada Y (2010) Pannexin 3 regulates intracellular ATP/cAMP levels and promotes chondrocyte differentiation. J Biol Chem 285:18948–18958
Ishikawa M, Iwamoto T, Fukumoto S, Yamada Y (2014) Pannexin 3 inhibits proliferation of osteoprogenitor cells by regulating Wnt and p21 signaling. J Biol Chem 289:2839–2851
Palumbo C (1986) A three-dimensional ultrastructural study of osteoid–osteocytes in the tibia of chick embryos. Cell Tissue Res 246:125–131
Ishihara Y, Sugawara Y, Kamioka H, Kawanabe N, Kurosaka H, Naruse K, Yamashiro T (2012) In situ imaging of the autonomous intracellular Ca2+ oscillations of osteoblasts and osteocytes in bone. Bone (NY) 50:842–852
Kato Y, Windle JJ, Koop BA, Mundy GR, Bonewald LF (1997) Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res 12:2014–2023
Kurata K, Heino TJ, Higaki H, Väänänen HK (2006) Bone marrow cell differentiation induced by mechanically damaged osteocytes in 3D gel-embedded culture. J Bone Miner Res 21:616–625
Adachi T, Aonuma Y, Tanaka M, Hojo M, Takano-Yamamoto T, Kamioka H (2009) Calcium response in single osteocytes to locally applied mechanical stimulus: differences in cell process and cell body. J Biomech 42:1989–1995
Mc Garrigle MJ, Mullen CA, Haugh MG, Voisin MC, McNamara LM (2016) Osteocyte differentiation and the formation of an interconnected cellular network in vitro. Eur Cell Mater 31:323–340
Noble BS (2008) The osteocyte lineage. Arch Biochem Biophys 2:106–111
Jing D, Baik AD, Lu XL, Zhou B, Lai X, Wang L, Luo E, Guo XE (2014) In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading. FASEB J 28:1582–1592
Adachi T, Aonuma Y, Ito S, Tanaka M, Hojo M, Takano-Yamamoto T, Kamioka H (2009) Osteocyte calcium signaling response to bone matrix deformation. J Biomech 42:2507–2512
Hoebe RA, Van Oven CH, Gadella TWJ, Dhonukshe PB, Van Noorden CJF, Manders EMM (2007) Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging. Nat Biotechnol 25:249–253
Woo SM, Rosser J, Dusevich V, Kalajzic I, Bonewald LF (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
Vazquez M, Evans BA, Riccardi D, Evans SL, Ralphs JR, Dillingham CM, Mason DJ (2014) A new method to investigate how mechanical loading of osteocytes controls osteoblasts. Front Endocrinol (Lausanne) 5:208
Boukhechba F, Balaguer T, Michiels JF, Ackermann K, Quincey D, Bouler JM, Pyerin W, Carle GF, Rochet N (2009) Human primary osteocyte differentiation in a 3D culture system. J Bone Miner Res 24:1927–1935
Sugawara Y, Kamioka H, Ishihara Y, Fujisawa N, Kawanabe N, Yamashiro T (2013) The early mouse 3D osteocyte network in the presence and absence of mechanical loading. Bone (NY) 52:189–196
Toyosawa S, Oya K, Sato S, Ishida K (2012) Osteocyte and DMP1. Clin Calcium 22:713–720
Schiavi SC (2006) Bone talk. Nat Genet 38:1230–1231
Atkins GJ, Rowe PS, Lim HP, Welldon KJ, Ormsby R, Wijenayaka AR, Zelenchuk L, Evdokiou A, Findlay DM (2011) Sclerostin is a locally acting regulator of late-osteoblast/preosteocyte differentiation and regulates mineralization through a MEPE-ASARM-dependent mechanism. J Bone Miner Res 26:1425–1436
Wang Z, Odagaki N, Tanaka T, Hashimoto M, Nakamura M, Hayano S, Ishihara Y, Kawanabe N, Kamioka H (2016) Alternation in the gap-junctional intercellular communication capacity during the maturation of osteocytes in the embryonic chick calvaria. Bone (NY) 91:20–29
Vanden AF, Bidaux G, Gordienko D, Beck B, Panchin YV, Baranova AV, Ivanov DV, Skryma R, Prevarskaya N (2006) Functional implications of calcium permeability of the channel formed by pannexin 1. J Cell Biol 174:535–546
This work was performed in part under the Cooperative Research Program of Institute for Frontier Medical Sciences, Kyoto University, Japan, and supported by a Grant-in-Aid for Scientific Research (No. JP 16H05549) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
Conflict of interest
All authors have no conflicts of interest.
About this article
Cite this article
Tanaka, T., Hoshijima, M., Sunaga, J. et al. Analysis of Ca2+ response of osteocyte network by three-dimensional time-lapse imaging in living bone. J Bone Miner Metab 36, 519–528 (2018). https://doi.org/10.1007/s00774-017-0868-x
- Fluid shear stress
- 3D time-lapse imaging