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Osteocyte Calcium Signals Encode Strain Magnitude and Loading Osteocyte calcium signals encode strain magnitude and loading frequency in vivo Karl J. Lewisa, Dorra Frikha-Benayeda, Joyce Louiea, Samuel Stephena, David C. Sprayb, Mia M. Thic, Zeynep Seref-Ferlengezc, Robert J. Majeskaa, Sheldon Weinbauma,1, and Mitchell B. Schafflera,1 aDepartment of Biomedical Engineering, City College of New York, New York, NY 10031; bDepartment of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461; and cDepartment of Orthopaedic Surgery, Albert Einstein College of Medicine, Bronx, NY 10461 Contributed by Sheldon Weinbaum, September 14, 2017 (sent for review May 11, 2017; reviewed by Tamara Alliston and Henry J. Donahue) Osteocytes are considered to be the major mechanosensory cells their environment. It also accounts for solute transport to and of bone, but how osteocytes in vivo process, perceive, and from osteocytes, including metabolites as well as key osteocyte- respond to mechanical loading remains poorly understood. Intra- produced signaling molecules such as sclerostin, RANKL, and cellular calcium (Ca2+) signaling resulting from mechanical stimu- OPG. Osteocyte expression of these key signaling molecules is lation has been widely studied in osteocytes in vitro and in bone sensitive to changes in mechanical load as well as other stim- explants, but has yet to be examined in vivo. This is achieved uli that initiate bone remodeling, such as changes in gonadal herein by using a three-point bending device which is capable of steroids and local tissue microdamage (1–4). delivering well-defined mechanical loads to metatarsal bones of Mechanical loads at the bone tissue level are translated to living mice while simultaneously monitoring the intracellular Ca2+ fluid flow in the LCS (5–7). Indeed, a growing body of evidence responses of individual osteocytes by using a genetically encoded indicates that fluid flow through the LCS dominates the local fluorescent Ca2+ indicator. Osteocyte responses are imaged by mechanical input to osteocytes in situ (8–11). Mechanical stim- using multiphoton fluorescence microscopy. We investigated the ulation of osteocytes increases cytoplasmic Ca2+, a ubiquitous in vivo responses of osteocytes to strains ranging from 250 to response to both mechanical and biochemical stimuli (12–15). 3,000 µ and frequencies from 0.5 to 2 Hz, which are character- In particular, Thi et al. demonstrated with cells that fluid forces istic of physiological conditions reported for bone. At all load- acting on the αvβ3 integrin adhesion sites on osteocyte cell pro- ing frequencies examined, the number of responding osteocytes cesses in vitro triggered Ca2+ responses that spread toward the 2+ increased strongly with applied strain magnitude. However, Ca cell body and then initiated a typical whole-cell Ca2+ signaling intensity within responding osteocytes did not change signifi- response like that seen in other cells (16). Finally, most studies cantly with physiological loading magnitudes. Our studies offer of osteocyte Ca2+ signaling have been conducted either in 2D a glimpse into how these critical bone cells respond to mechanical cell cultures or in explanted bones (10, 17), which have limita- load in vivo, as well as provide a technique to determine how the tions. Cell cultures lack the LCS and attendant localized tissue cells encode magnitude and frequency of loading. architecture and fluid flow, while bone explants are subject to postmortem tissue changes, as well as fluid pressure loss in the osteocytes j calcium signaling j mechanotransduction j LCS resulting from loss of in vivo vascular pressure (8, 18, 19). in vivo loading j bone In the present studies, we report an in vivo approach to explore osteocyte Ca2+ signaling in whole bone where the cells one has a remarkable ability to sense mechanical load and can be individually imaged during mechanical loading. We gen- Badapt its structure in response. This mechanical adaptation erated a mouse model with an osteocyte-targeted genetically ability is so prominent for bone that it was among the first organ encoded Ca2+ indicator (GECI) to study Ca2+ signaling in osteo- systems to be recognized in this regard; indeed, the word “ortho- cytes in their authentic in vivo environment. We also created a pedics,” first coined in the mid-1700s, derives from straightening bone loading approach for use in live mice while simultaneously the bones of children. The ability of bone to adapt its structure is essential for growing a skeleton appropriate to its mechani- Significance cal demands and for maintaining the integrity of that skeleton throughout life. Osteoblasts and osteoclasts are the effector cells that carry out bone formation and resorption (respectively), and Osteocytes are thought to be the major regulator of bone their dysfunction leads to growth defects and osteoporosis. How- mechanosensation events. However, little is known about ever, it is increasingly recognized that osteocytes, buried within how osteocytes in vivo acutely respond to tissue-level the bone matrix, are the major local orchestrators of diverse mechanical loading. We report here a technique for the direct functions in bone. They produce soluble signaling factors that in vivo observation of osteocyte calcium signaling events with regulate both bone formation and resorption, local mineraliza- simultaneous whole bone loading. Our results demonstrate tion, and also calcium (Ca2+) and phosphate transport at the that osteocyte populations integrate mechanical signals by bone matrix level. They also function as endocrine cells, produc- altering the number of responding cells and that this effect ing factors such as FGF23, that have an impact on renal func- is dependent on loading frequency. tions. Among the most essential functions of osteocytes is sens- Author contributions: K.J.L., D.C.S., S.W., and M.B.S. designed research; K.J.L., D.F.-B., ing mechanical load to which bone is subjected and transducing M.M.T., and Z.S.-F. performed research; K.J.L., J.L., and S.S. analyzed data; and K.J.L., ENGINEERING this load into downstream signals, which regulate osteoblasts and R.J.M., and M.B.S. wrote the paper. osteoclasts. Reviewers: T.A., University of California, San Francisco; and H.J.D., Virginia Common- Osteocytes in situ exist in a complex, 3D, highly intercon- wealth University. nected array. Osteocyte cell bodies and processes reside in The authors declare no conflict of interest. spaces (lacunae and canaliculi, respectively) within bone matrix Published under the PNAS license. and are surrounded in these spaces by a fluid-gel layer that 1To whom correspondence may be addressed. Email: [email protected] or fills the lacunar-canalicular space (LCS). This fluid layer, which mschaffl[email protected]. PHYSIOLOGY moves when bones are mechanically loaded, transmits mechan- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ical forces and determines much of how osteocytes experience 1073/pnas.1707863114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1707863114 PNAS j October 31, 2017 j vol. 114 j no. 44 j 11775–11780 Downloaded by guest on September 26, 2021 ing off-axis stresses. MT3 bones from OtGP3 mice had larger cortical bone areas, but slightly lower tissue mineral density than WT bones (Table 1; P < 0.05), resulting in similar cross-sectional moduli and diaphyseal bending rigidities. Osteocyte Response to Mechanical Loading in Vivo. We used a custom-designed device to deliver controlled mechanical strains to MT3 bones of living OtGP3 mice while simultaneously moni- toring intracellular Ca2+ responses with MPM. Ca2+ fluorescence in responding cells increased very rapidly, within 200 ms of load- ing onset (Fig. 2), and oscillations followed the applied loading (Movie S1). Osteocytes were examined in bones loaded cycli- cally to habitual physiological strain magnitudes [250, 500, 1,000, and 2,000 microstrain(µ)] as well as a nonhabitual high strain level (3,000 µ). Loading frequency effects were examined as well Fig. 1. Multiphoton z-stack image of OtGP3 MT3 osteocytes in vivo. Note (0.5, 1, or 2 Hz; n = 6 animals per loading frequency group). that all cells in the field of view exhibit GCaMP3 signal, indicating highly Once activated, Ca2+ response intensity remained consistent efficient expression in osteocytes. for each osteocyte for loading up to 2,000 µ. Osteocyte Ca2+ intensity increased only with loading to 3,000 µ (Fig. 3; P < 0.05, ANOVA). In contrast, the number of Ca2+ responding osteo- observing the intracellular Ca2+ responses of individual osteo- 2+ cytes increased markedly with increasing strain magnitude for cytes with multiphoton microscopy (MPM). We report how Ca all loading frequencies tested (Fig. 4). At 0.5- and 1-Hz loading, signaling of osteocytes in vivo, both individually and as networks, the load–response curves (i.e., relationship between responding responds to mechanical loading. We used this approach to test osteocytes and strain magnitude) were linear (P < 0.01). How- the hypothesis that encoding of mechanical strain and frequency ever, osteocyte “recruitment rate” (i.e., the number of respond- information by osteocytes in vivo is based on recruitment of ing osteocytes per strain level) was approximately twofold responding cells (i.e., numerical encoding) rather than on graded greater at 1- vs. 0.5-Hz loading (0.023 vs. 0.012; P < 0.001, anal- 2+ Ca responses by individual cells. ysis of covariance). The load–response curve was fundamentally different at 2 Hz. At this frequency, the number of Ca2+ respond- Results ing osteocytes was near zero for strains of <1,000 µ. Above this Osteocyte GCaMP3 Expression. Mice expressing the osteocyte- strain level, osteocyte recruitment increased steeply. The rela- targeted GECI GCaMP3, which are here termed OtGP3, were tionship between applied strain levels and responding osteocytes created. Baseline GCaMP3 fluorescence was visible in osteo- at 2-Hz loading appeared nominally exponential, but was better cytes throughout the diaphyseal cortex (Fig. 1). In pilot stud- described by a polynomial relationship (r2 = 0.55 vs.
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