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Reviewers: and paper. the J.L., wrote K.J.L., M.B.S. and research; R.J.M., performed Z.S.-F. and M.M.T., simultaneously a while created mice live also in We use environment. for vivo approach loading in bone authentic genetically their osteocyte-targeted in an cytes with Ca model gen- encoded We mouse loading. a mechanical during erated imaged individually be can Ca 19). osteocyte 18, explore (8, the pressure in vascular loss vivo to in pressure of subject fluid loss as from are resulting well explants LCS as bone tissue changes, localized while tissue limita- attendant flow, postmortem have and fluid which LCS and 17), the architecture lack (10, cultures bones Cell explanted tions. in studies or most cultures Finally, cell (16). 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PHYSIOLOGY ENGINEERING 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. 0.85, respec- ies, we found that >95% of osteocytes in OtGP3 mice exhib- tively). Responding osteocytes were distributed within regions ited detectable fluorescence. Fluorescence intensity level varied of interest (ROIs) without an apparent spatial pattern. We among cells, and resting osteocytes exhibited low-level fluctua- 2+ observed instances in which loading at a given strain level trig- tions in Ca fluorescence intensity (Fig. 2). gered a Ca2+ response in one osteocyte, but none in an adjacent osteocyte. Bone Structure in OtGP3 Mice. Our studies used the third meta- To address the importance of vascular pressure to maintaining tarsal (MT3) of the mouse foot. Previous studies in our group the fluid pressure of the LCS, MT3s from an additional group of demonstrated that this bone is well-suited for direct visualiza- mice (n = 3) were cyclically loaded to a single test strain (2,000 tion of cortical bone osteocytes in living animals by using MPM µ, 1 Hz) for 60 s while animals were alive and then again at (20). Its tubular geometry lends itself to bending without creat- 15 min postmortem (no vascular pressure but cells viable) and 60 min postmortem (severe mitochondrial stress) (20). Ampli- tude of osteocyte Ca2+ response was attenuated markedly at 15 min after death compared with in vivo loading (Fig. 5). These small Ca2+ oscillations in response to loading became irregular and did not track the applied loading frequency. The situation further decayed by 60 min. Discussion This study provides a report of in vivo osteocyte Ca2+ responses to mechanical loading of bone in living animals and provides

Table 1. Bone structure, mineralization, stiffness, and osteocyte density in WT and OtGP3 MT3 bone Parameter B6-WT OtGP3

Bone length, mm 7.75 ± 0.07 7.59 ± 0.14* Total bone area, mm2 0.208 ± 0.25 E-2 0.205 ± 0.12 E-2 Fig. 2. A representative Ca2+ trace for a single osteocyte before (dashed TMD, g/cm3 1.15 ± 0.02 1.05 ± 0.02* line) and after the start of cyclical loading (solid line) at 1 Hz to 3,000 µ. Sectional stiffness, N/m 7.58 ± 0.36 7.82 ± 1.03 2+ Before mechanical loading, all osteocytes exhibited low-level Ca fluctu- Ot.N, no./mm2 491 ± 38 448 ± 39* ations. Immediately following the start of mechanical loading, responsive cells showed cyclic increases in Ca2+ fluorescence intensity coinciding with Values are mean ± SD. n = 6 per group. Ot.N, osteocyte number; TMD, the applied loading frequency. tissue mineral density. *P < 0.05.

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(n dine which and (53) cross-sections, area middiaphyseal elsewhere bone thick detailed woven mm as per vs. (number bones Lamellar sity C57Bl/6 genotype). (µCT) WT micro-CT per using vs. bones by OtGP3 performed assess Bones. were Metatarsal to studies B6 mineralization and and OtGP3 of tural of Properties College Material and City Structural and Medicine of College Com- Use Einstein all York. and Albert and New Care both mice Animal female Institutional at the 16-wk-old mittees by used approved Studies were bred is background. were procedures which Mice promoter, C57BL/6 (52). Cre DMP1 a odontoblasts the and onto possess of osteocytes in fragment which expressed 10-kb dominantly a Labs], by driven JAX recombinase [B6N.FVB-Tg(Dmp1-cre)1Jqfe/BwdJ; mice Ai38 mice mating DMP1/Cre by with Labs] obtained JAX were [B6;129S-Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J; OtGP3) (designated GCaMP3. GCaMP3 targeted Osteocyte-Targeted Expressing Mice in found be can Methods. methods and study Materials and SI development device regarding Details Methods and Materials osteoporosis. as such predic- diseases even or skeletal characteristic of be tive may recruit- input osteocyte mechanical between and couplings ment the disrupt that alterations ei tal. et Lewis .Vrrge W aga J caaaL 21)Fudflwi h osteocyte the in flow Fluid (2013) Flow-induced LM bone: McNamara in TJ, transport Vaughan solute SW, and Verbruggen Fluid 9. (2009) S Weinbaum orches- SP, Master Fritton Osteocytes: (2013) 8. O Kennedy RJ, signaling. Majeska WY, Wnt Cheung and MB, Schaffler mechanosensing Osteocytes, 7. (2008) ML of Johnson role LF, the Bonewald for model 6. A (2007) S Weinbaum MB, Schaffler LM, McNamara Y, involves Wang bone 5. fatigue-loaded in resorption of Activation (2012) al. et follow- OD, resorption Kennedy bone of control 4. and apoptosis Osteocyte (2010) al. et KB, in Emerton increase 3. the prevents apoptosis osteocyte of Inhibition (2015) al. et LI, Plotkin 2. unload- Hindlimb by caused apoptosis Osteocyte (2016) al. et P, Cabahug-Zuckerman 1. sectswr iulzdi middiaphyses in visualized were Osteocytes Methods. and Materials SI inadtasuto nosteocytes. in transduction and tion loading. mechanical dynamic under bones 1592. long mouse intact approach. interaction fluid–structure Mechanobiol A environment: mechanical mechanotransduction. bone. of trators Bone osteocytes. in 104:15941–15946. mechanotransduction induced flow in integrins popu- osteocyte distinct by signaling lations. pro-osteoclastogenic active and apoptosis both mice. in ovariectomy ing unloading. by induced bone of loss 18942. the or resorption factor bone nuclear of activator receptor osteocytic of resorption femurs. mice subsequent in and bone production Trabecular RANKL and osteocyte cortical trigger to required is ing tayadoclaoyfli flow. fluid oscillatory and steady tan pto up Strains µ. 42:606–615. Bone 50:1115–1122. 13:85–97. acfTsu Int Tissue Calcif r presented are S3 ) (Fig. data calibrations strain and S2) Fig. 2 eemaue t400× at measured were ) nuRvFudMech Fluid Rev Annu ∼2,000 Bone 46:577–583. 94:5–24. µ Bone Bone cu ndahssi ioadaecharac- are and vivo in diaphyses in occur 51:466–473. 54:182–190. ecetdamcaia loading mechanical a created We 41:347–374. oeMnrRes Miner Bone J iad(AK)btde o stop not does but (RANKL) ligand κB ieehbtn osteocyte- exhibiting Mice anfiainfo 5-µm- from magnification stpclo extreme of typical is µ) 2+ ne sflrn anes- isoflurane Under inln 5) Bones (54). signaling ilChem Biol J rcNt cdSiUSA Sci Acad Natl Proc 31:1356–1365. AE J FASEB imc Model Biomech 290:18934– 28:1582– Struc- 6 = 3 aosC,Tmysti ,Csil B(00 sect ehnbooyadpericel- and signal- mechanobiology Osteocyte calcium (2010) AB Castillo intracellular S, Temiyasathit of CR, Jacobs properties 23. Spatiotemporal (2013) al. et D, Jing 22. mediates mechanism ATP-dependent An (2010) XE Guo Q, Xu KD, Costa XL, Lu B, differ- Huo Regional 21. (2016) MB Schaffler RJ, Majeska space J, pore Basta-Pljakic hierarchical D, the in Frikha-Benayed flow interstitial 20. and Blood (2015) L venous Cardoso to induced due SC, adaptation Cowin flow bone On 19. fluid (2003) SC Dynamic Cowin S, (2015) Weinbaum SP, YX Fritton L, Qin Wang J, 18. Jiao DE, Gibbons GW, Tian M, Hu Mechanosen- 17. (2013) DC Spray S, Weinbaum MB, and Schaffler SO, oxide Suadicani nitric MM, of Thi production 16. The (2001) J Klein-Nulend K, Soejima by AD, production prostaglandin Bakker increases 15. flow fluid Pulsating (1996) al. et NE, Ajubi extra- 14. and stores Ca2+ Intracellular (1996) CT Brighton SR, Pollack FD, Allen CT, Hung 13. ihFg .Ti okspotdb I rnsA011,AR057139, AR041210, Grants NIH by supported work assistance for This DK081435. and Fritton 6. DK091466, Susanah Dr. Fig. and with microscopy multiphoton with tance ACKNOWLEDGMENTS. IBM). 20; (Version SPSS anal- with regression performed or ANOVA testing were either hoc Analyses post using yses. for by difference examined squared were least Fisher’s groups with strain among level the strain using vs. by assessed were strains Analyses. Statistical exhibited they loading. during if intensity responding fluorescence normalized as Accord- in 60). considered increase (59, intensity were fluorescent osteocytes in signal- change ingly, lower fold a potential have lower a GECIs thus, other Ca period and exogenous 30-s than GCaMP3 a ratio loading. for to-noise intensity of mean start its the to normalized before was Fluorescence cell (NIH). each ImageJ for using intensity images postprocessing by performed were Ca ROI. the within located tion plane 6 optical single at a acquisition in ∼20 imaged filter were bandpass 40× Osteocytes 560-nm second. a per to frames using 490- 920-nm By with and performed MPM. excitation was wavelength using imaging objective, by immersion visualized water nification was loading bone whole death; studies Imaging. after explant min bone 60 in and reported 15 times 58). 17, at incubation (10, repeated approximates were min imaging 60 and loading and Ca additional from cyte MT3 vivo. in (n response mice osteocyte to contributes pressure lar study. loading the of euthanized end were imag- the Mice from observed. at adjacent response was the osteocyte effect in sequential one at No offset at strain sequence. or loading conditioning lowest strain potential to highest assess to highest to site from lowest ing then from was and going procedure site level, This imaging (57). strain cells each bone min, for 15 for for repeated period rest refractory to allowed the and at exceeds unloaded period then which loading was 60-s bone The a strain. for test continuously each collected were strain (see Images measurable studies details). no for calibration effectively strain had on they based because gradients middiaphysis. chosen the of were side ROIs either on These (n immediately Hz located 2 ROIs two or in 1, acquired 0.5, at tested groups with well, nadto,w sdti xeietlsse oass hte vascu- whether assess to system experimental this used we addition, In n notoyi n sebatccl ewrsudrfli flow. fluid mechanics. lular under networks cell osteoblastic and 540. osteocytic in ing nanoindenta- cell single under network cell bone tion. in signaling calcium intercellular osteo- bone cortical among activity cytes. mitochondrial and metabolism oxidative in ences tissue. bone of architecture stasis. oscillations. calcium Biophys osteocyte situ and in of modulation processes mechanobiological along occur require forces and physiological 21017. body to cell osteocytes not of responses dependent. stress sory shear is cells bone primary by 671–677. 2 E prostaglandin Commun process. cytoskeleton-dependent osteocytes–a chicken cultured experiencing cells bone of response Ca2+ real-time flow. the fluid in required are Ca2+ cellular eo h eiselsraet ananuiomsri distribu- strain uniform maintain to surface periosteal the below µm elCalcium Cell 2+ ae)wr oddt igets tan(2,000 strain test single a to loaded were males) 3 = Bone Biomech J sect Ca Osteocyte 579:55–61. epnewsmaue.Mc eete uhnzdi place, in euthanized then were Mice measured. was response 225:62–68. Biomech J 90:15–22. nuRvBoe Eng Biomed Rev Annu 36:1439–1451. 47:234–241. PNAS The 29:1411–1417. 2+ 2+ etakD.Ara orge-otea o assis- for Rodriguez-Contreras Adrian Dr. thank We Tadhsooia ifrnebtenmouse between difference histological and µCT nest esrmnsfo sectsobserved osteocytes from measurements intensity | epnei T oe fOG3mc during mice OtGP3 of bones MT3 in response Biomech J α coe 1 2017 31, October Vβ integrin. 3 2+ t esn ys( s 2 epciey,and, respectively), 12, vs. (5 dyes sensing 48:842–854. et ifrne notoyeresponse osteocyte in Differences test. 12:369–400. rcNt cdSiUSA Sci Acad Natl Proc e rqec) mgswere Images frequency). per 6 = | o.114 vol. IMtrasadMethods and Materials SI | ice ipy Res Biophys Biochem o 44 no. ,adosteo- and µ), Biomech J Bone rhBiochem Arch 110:21012– | 53:531– >25% 11779 mag- 34:

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