Bone 54 (2013) 182–190

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Review

Mechanosensation and transduction in osteocytes

Jenneke Klein-Nulend a,⁎, Astrid D. Bakker a,1, Rommel G. Bacabac b, Aviral Vatsa c, Sheldon Weinbaum d

a Department of Oral Cell Biology, ACTA-VU University Amsterdam, Research Institute MOVE, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlandsb Department of Physics, Medical Biophysics Group, University of San Carlos Talamban Campus, Cebu City, Philippinesc Department of Regenerative Medicine, School of Science Technology and Health, University Campus Suffolk, James Heir Building, Neptune Quay, Ipswich, IP4 1QJ, UKd Department of Biomedical Engineering, The City College of New York, New York, NY 10031, USA

⁎ Corresponding author. Fax: +31 20 5980333.E-mail addresses: j.kleinnulend@acta.nl (J. Klein-Nu

(A.D. Bakker), rgbacabac@gmail.com (R.G. Bacabac), aviweinbaum@ccny.cuny.edu (S. Weinbaum).

1 Fax: +31 20 5980333.

8756-3282/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.bone.2012.10.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 July 2012Revised 9 October 2012Accepted 10 October 2012Available online 18 October 2012

Edited by: Lynda F. Bonewald, Mark E. Johnson,and Michaela Kneissel

Keywords:MechanosensationMechanotransductionOsteocytesOsteoblastsOsteoclastsFluid flow

The human skeleton is a miracle of engineering, combining both toughness and light weight. It does so becausebones possess cellular mechanisms wherein external mechanical loads are sensed. These mechanical loads aretransformed into biological signals, which ultimately direct bone formation and/or bone resorption. Osteocytes,since they are ubiquitous in the mineralized matrix, are the cells that sense mechanical loads and transduce themechanical signals into a chemical response. The osteocytes then release signalingmolecules, which orchestratethe recruitment and activity of osteoblasts or osteoclasts, resulting in the adaptation of bonemass and structure.In this review,wehighlight current insights in bone adaptation to externalmechanical loading,with an emphasison how amechanical load placed on whole bones is translated and amplified into a mechanical signal that issubsequently sensed by the osteocytes.This article is part of a Special Issue entitled "The Osteocyte".

© 2012 Elsevier Inc. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182Which mechanical signal activates the osteocytes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183How do osteocytes perceive the mechanical signal? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185What constitutes the osteocyte response to mechanical stimulation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

Introduction

Bone mass and architecture are affected by external mechanicalloads exerted during daily physical activity (Fig. 1). Adaptation ofbone mass and structure is achieved during a process of repeatedturnover by bone cells under influence of mechanical stimuli. Theprinciple that functional adaptation of bone is the end result of a

lend), a.bakker@acta.nlralvatsa@gmail.com (A. Vatsa),

rights reserved.

self-organized (bone) cellular process was to a large extent recognizedby William Roux, as early as 1881 [1]. However, it was not until morethan a century later, when the isolation of that elusive cell calledthe osteocyte became possible, that the central role of the osteocytesin the process of mechanical adaptation was recognized.

Osteocytes express, among other proteins, osteocalcin, osteonectin,and osteopontin, but show little alkaline phosphatase activity, particularlythemoremature cells. Although thesemarkers are typically expressed byosteocytes, they are not specific for them. For a long time, no osteocytespecific markers were known. This changed when monoclonal antibodyMAb OB7.3 was developed by the group of Nijweide [2]. MAb OB7.3 isspecific for avian osteocytes and found to be the avian homologue ofmammalian Phex [3]. The antibody allowed the isolation of an almost

Fig. 1. Cross section through the head of a femur clearly showing the alignment of thetrabeculae (rectangle) along the direction of principle loading (arrow), which islongitudinal.

Fig. 2. Immunofluorescence image of phalloidin-labelled actin cytoskeleton in osteocytesin situ inmouse calvaria. Approximately 50–60 cell processes (arrowhead) originate fromeach osteocyte cell body (arrow) and radiate through the canaliculi, in different directionsto form an intercellular network with surrounding osteocytes. Bar, 15 μm.

Fig. 3. A schematic sketch representing the central role of osteocytes in boneremodelling. Osteocytes sense the mechanical load and subsequently produce signalingmolecules like nitric oxide and prostaglandins that can regulate the activity of the effectorcells, the osteoclasts and the osteoblasts,which subsequently leads to adequate bonemassand architecture. OB, osteoblast; OCL, osteoclast.

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>95% pure population of osteocytes from calvariae of 18-day-old chickenfetuses using immunomagnetic separation [2], and the study of character-istics and properties of these osteocytes [4]. Using this antibody it wasshown for the first time that isolated osteocytes are much more respon-sive tomechanical load in the formof pulsatingfluidflow thanosteoblastsor periosteal fibroblasts [5].

Osteocytes are the pivotal cells orchestrating the biomechanicalregulation of bone mass and structure for efficient load bearing [5–9].The mechanosensitive osteocytes comprise 90-95% of the whole bonecell population in the adult animal [10]. Within the hard mineralizedmatrix, osteocyte cell bodies reside in the spaces called the lacunae.From each osteocyte cell body, approximately 50–60 cell processes orig-inate and radiate through the mineralized matrix via spaces called thecanaliculi. Together these structures are called the lacuno-canalicularsystem (LCS). These cell processes radiate in different directions andform an intricate intercellular network of osteocytes (Fig. 2), which isdirectly connected to the cells lining the bone surface and cells withinthe bone marrow [11–13]. How the osteocytes sense the mechanicalloads on bone and coordinate adaptive alterations in bone massand architecture is not yet completely understood. However, it iswidely accepted that mechanical loads placed on bones as an organdrive a flow of interstitial fluid through the unmineralized pericellularmatrix surrounding osteocytes and their dendritic processes [9,14].This flow is then thought to somehow activate the osteocytes, whichproduce signalingmolecules that can regulate the activity of the effectorcells [15,16], the osteoclasts and the osteoblasts, leading to adequatebone mass and architecture [17] (Fig. 3).

Over the past two decades theoretical and experimental studies havecontributed in delineating the role of osteocytes in mechanosensationand their subsequent biological response. New insights have emergedfroman enhanced understanding of the anatomical details of the primaryosteocytes [4,11,13,18], osteocyte isolation [2,19], mechanosensation[20], and signal transduction [21–24], to name just a few of theseadvances. Computational models have demonstrated the importanceof mechanical loading as a potent and stable regulator of complex bio-chemical processes involved in maintenance of bone architecture [17].If osteocytes, acting as the bone mechanosensors, indeed orchestratethe adaptation of bone to mechanical loading, the question arises how

this biological action is performed. Which mechanical signal activatesthe cells? How do the cells perceive the mechanical signal, and whatconstitutes the response? In other words, how do mechanosensitiveosteocytes supervise the process of bone remodeling, which is thecentral phenomenon in bonemass regulation in accordancewith externalmechanical loading?

Which mechanical signal activates the osteocytes?

Mechanical loading placed on bone can result in mechanosensationby osteocytes via several potential mechanisms, which were initiallyreviewed at the macroscopic level [7]. These mechanisms includedchanges in whole tissue strain, hydrostatic pressure, and streamingpotentials generated by bone fluid flow through a charged bonematrix.Streaming potentials were initially thought to be generated by electro-kinetic effects associatedwith a system of connectedmicropores associ-ated with the collagen-apatite porosity [25]. Subsequently, Cowin et al.[26] and Zhang et al. [27] proposed that the pores were actually thecanaliculi in the mineralized bone and these channels were the siteof the strain generated potentials. Those electrokinetic effects might

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modulate the movement of ions such as calcium across the cell mem-brane [28,29]. Load that is rapidly placed on bone first pressurizes theinterstitial fluid around the osteocytes, before the fluid is driven toflow. Zhang et al. [27] estimated that the fluid component could carryas much as 12% of the applied mechanical load and produce peak pres-sures of 2–3 MPa. More recently, Gardinier et al. [30] have predictedthat the magnitude of the pressure experienced by osteocytes in vivocould reach up to 5 MPa. Klein-Nulend et al. [5] subjected osteocytes,osteoblasts, and periosteal fibroblasts from chicken calvarial bone totwo different mechanical stimuli, i.e. hydrostatic compression (IHC)and pulsatile fluid flow (PFF). Osteocytes were particularly sensitiveto fluid shear stress, more so than to hydrostatic stress, although onecan argue that the hydrostatic pressure applied, i.e. 13 kPa, is muchlower than the 5 MPa predicted to occur in vivo [30]. More recent re-search has shown that cyclic hydraulic pressures of 68 kPa can modu-late signaling molecule production in cells of the mouse MLO-Y4osteocyte cell line [31]. Over the past decade a number of theoreticaland experimental studies have appeared that have put forth evidencestrongly favoring interstitial fluid flow and direct cell strain as opposedto streaming potentials or hydrostatic pressure as themost likelymech-anism for mechanosensation.

Osteocytes form a ‘network’ throughout the bone matrix byconnecting with each other and with surrounding lining cells on thebone surface. These anatomical characteristics of osteocytes make themideally placed in bone to sense external mechanical loads imparted onbone. Osteocytes are directly connected with each other via gapjunction-coupled long slender cell processes which run along the centralaxis of the canaliculi except where there are ridges created by transversecollagen fibrils. An intracellular as well as an extracellular route allow forrapid passage of ions and signal molecules, enabling cellular signalingfrom osteocytes deep within the bone tissue to surface lining cells andvice versa. The pericellular space is filled with a proteoglycan richmatrixwith tethering fibers that attach the process to the canalicular wall [32].Any mechanical loading-driven interstitial fluid flow through thepericellular space is dominated by this matrix since it controls both thehydraulic resistance and the size of the molecules that can be convectedfor nutritional needs.

Piekarski and Munro [14] were the first to propose that mechanicalloading induced fluid flow in bone and that this was necessary for bothnutrition and waste removal. Early models of an osteonal fluid flowneglected both the presence of the cell process and the pericellularmatrix in the canaliculi [33]. More refined models that consideredboth structures showed that the load-induced fluid flow was drivenradially inward from the cement line of an osteon toward the osteonalcanal, and that the relaxation time for this behavior matched wellwith the decay of streaming potentials when the molecular sieve forthematrixwas roughly the size of albumin (7 nm) [34]. This theoreticalpredictionwas confirmed byWang et al. [35]who delineated the bone'sinterstitial fluid pathway in vivo using tracers varying in size fromprocion red to ferritin. These studies emphasized the importance ofmechanically induced flow for the transport of metabolites to andfrom osteocytes in an osteon, to ensure osteocyte viability. Numeroustracer studies have been conducted, which are summarized by Frittonand Weinbaum [36]. These studies show that the size of the molecularsieve is slightly greater than horseradish peroxidase (~6 nm) [37,38],easily allows the passage of microperoxidase (~2 nm), and that asmall tracer, such as procion red (~1 nm), is confinedwithin the bound-aries of the LCS [37,35]. One can show using fibermatrix theory that thefluid shearing stresses on the cell processwould be 20–30 times greaterif this matrixwere not present. This is of great importance in comparingfluid shearing stresses in vivo and in culture studies.

While theoretical models have been used to predict fluid flow in theLCS due to mechanical loading it has been much more difficult to dem-onstrate this experimentally. Wang et al. [39] have developed a noveltechnique that combines fluorescence recovery after photobleaching(FRAP) with confocal microscopy to directly measure real time solute

movement in intact bones. In this technique, the movement of a vitallyinjected fluorescent dye between individual lacunae can be visualized insitu with laser scanning confocal microscopy. For unloaded bone one candetermine the diffusion coefficient offluorescein and determine from thismeasured value and the molecular size the mesh pore size of thepericellular matrix confirming the ~7 nm estimated from tracer studies.Su et al. [40] extended this approach to externally applied loading ofthe mouse femur and showed that the fluorescent recovery time wasreduced by ~25%. In the latest study Price et al. [41] combined FRAP ina mouse tibia with computational modeling and was able to predictthe peak computational fluid velocity during cyclic loading, 60 μm/s,and also estimate the peak resultant fluid shear stress ~5 Pa. These pre-dictions are based on a three compartment model which considers thepericellular matrix surrounding the osteocyte cell processes in theircanaliculi.

In the original fluid flow hypothesis [9] the activation of theosteocytes was proposed to be due to fluid shear stress acting on thecell process membrane. Numerous experimental studies were subse-quently conducted exposing bone cells in culture to steady and pulsatilewall shear stresses in the range 0.6 to 3.0 Pa predicted by the model in[9]. A typical in vitro study [42] is conducted in a two-dimensional (2D)environment on surface attached MLO-Y4 osteocyte-like cells asopposed to the three-dimensional (3D) in vivo environment of bonema-trix where the osteocyte morphology and pericellular flow environmentare different. There are several differences between the flow-induced ac-tivation of bone cells in vivo and in vitro. In vivo the cells are attached totheir mineralized matrix either through tethering filaments, or perhapsthrough integrin-based focal adhesions. β3 integrins have been observedon the cell processes and β1 integrins are found to be ubiquitous [43]. Invitro there is no pericellular matrix surrounding the cell and the attach-ments to the substrate are all integrin-based. The second difference isthe flow environment itself. As shown in [41] the fluid drag forces onthe pericellular matrix surrounding the cell process in vivo are 20-foldthose of the fluid shear stress acting on the cell process membrane. Invitro the fluid shear stress in nearly all experiments is the same on thecell processes and the cell body. This raises the important issue, whichpart of the osteocyte is its mechanosensing organelle, its process or itscell body, which we discuss in the next paragraph. Third, osteocytesseeded on a flat, stiff surface spread out and build up strong basal attach-ments to their substrate. It has been shown that round non-adherentosteocytes are an order of magnitude more sensitive to a mechanicalstimulus than a flat adherent osteocyte [44]. The mechanosensitivity ofosteocyteswith amore 3Dmorphology, such as occurs in vivo, may thusdiffer from that of adherent osteocytes. In summary, experiments withosteocytes cultured in 2D on flat surfaces may not suffice to unravel theintricate mechanisms used by osteocytes to transduce a mechanicalsignal into a chemical response. However, in vitro experiments un-doubtedly do provide valuable insights into which signaling moleculesare produced by osteocytes in response to a mechanical stimulus. Forexample, in vitro experiments illustrate that Wnt/β-catenin targetgenes are upregulated whereas sclerostin is downregulated in bonecells in response to mechanical stimulation which corresponds directlyto the in vivo situation [45–48]. In addition, the fact that osteocytesproduce factors that stimulate osteoclast formation in the absenceof mechanical loading, but not after being subjected to a mechanicalstimulus, was confirmed both in vitro [49] and in vivo [6].

Despite the differences in flow-induced mechanical loading in vivoand in vitro already discussed, there have been several in vitro studiesthat attempted to decipher which part of the cell, its process or thecell body, is more sensitive to mechanical forces. Adachi et al. [50]used a glass microneedle to apply separate local deformations onthe osteocyte process and cell body. They observed that a significantlylarger deformation was necessary at the cell body to induce a calciumresponse, and concluded that mechanosensitivity of the processes washigher than that of the cell body. Recent findings by Burra et al. [51],where theymanaged to differentially stimulate osteocyte cell processes

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and body using a transwell system, show that integrin attachmentsalong the cell processes act asmechanotransducers. Subsequent studiesby Litzenberger et al. [52] demonstrated that PGE2 release is mediatedby a β1 integrin. Most recently, Wu et al. [53] have developed a novelStokesian fluid stimulus probe to focally apply pN level hydrodynamicforces on either the osteocyte cell processes or body. Strikingly, large in-creases in electrical conductance were observed only when the pipettetip was directed at local integrin attachment sites along the process butnot on the cell body or on portions of the process thatwere not attachedto the substrate. This new approach clearly demonstrated that forcesbetween 1 and 10 pN could open stretch activated ion channels alongthe process at points of integrin attachment. These forces were of thesame magnitude as the forces predicted for the integrin attachmentsin vivo resulting from flow-induced mechanical loading [20].

How do osteocytes perceive the mechanical signal?

Osteocytes have a typical stellatemorphology and cytoskeletal organi-zation, which is important for the osteocyte's response to loading [54].The actin cytoskeletal structure differs greatly between the processesand the cell body, the former comprised of prominent actin bundlescross-linked by fimbrin [55] and the latter comprised of anti-parallelactin filaments cross-linked by α-actinin. This leads to a structurewhere the cell process has been estimated to be several hundred timesstiffer than the cell body [56]. This structure is retained after their isolationfrom bone [55] and is central to the transfer of mechanical forces.

Osteocytes are the descendants of osteoblasts, and similaritieswould be expected of cells of the same lineage. Yet these cells havedistinct differences, particularly in their responses to mechanicalloading and utilization of the various biochemical pathways to accom-plish their respective functions [57]. The differences between markers,morphology, and function of the polygonal matrix producing osteoblastand the embedded dendritic osteocyte are dramatic, yet the practice ofusing osteoblast cell lines to represent osteocytes continues mainlybecause of availability and ease of use. The field would benefit from thegeneration of a cell line with the properties and function of the matureosteocyte.

The prevalent, widely accepted hypothesis aboutmechanosensationby osteocytes proposes that the osteocyte cell processes lie at the heartof mechanosensation. Based on a 2D, surface-attached MC3T3-E1 cellstudy, it is believed that the fluid flow-mediated shear forces in thelacunae are too low to be sensed by the osteocyte cell bodies [58].However, substrate deformation (direct matrix strains) in vivo mightbe sufficient inmagnitude to affect osteocyte cell bodies [59]. Moreover,it has been shown that the osteocyte cell bodies respond in anintegrin-dependent manner after mechanical perturbation of the cellbody alone, showing that osteocyte cell bodies, in principle, aremechanosensitive [60]. Finally, the relative flat and spread shape of iso-lated osteocytes in 2D culture may greatly hamper their sensitivity to amechanical stimulus [45], and strains that are not able to elicit a re-sponse in bone cells adhered to a flat and stiff surface may be perfectlyable to elicit a response in cells in their natural 3D conformation. This issuggested by the fact that bone cells with rounded cell bodies appear tobe more mechanosensitive than cells that are less firmly attached, asnoted earlier. The osteocyte cell bodies in vivomay thus be involved indirect mechanosensation of matrix strains via their cytoskeleton.

The 3D shape and orientation of the long axes of osteocytes differin situ in two types of bone, fibula and calvaria, which have differentmechanical loading patterns. These clear differences in osteocytemorphology and alignment are possibly attributed to the fact thatthe external mechanical forces influence cytoskeletal structure andthus cell shape [61]. Indeed the fibula, which is predominantlyunidirectionaly-loaded, contains osteocytes with chiefly unidirectionalorientation of their long axes, and the calvaria, which are loaded radiallydue to intracranial pressure and/or mastication, contain osteocyteswhich are relatively randomly oriented [61]. In addition, cells in culture

align due to integrin-mediated elongation of stress fibers in the direc-tion of principle strains [62,63]. The internal organization of the cellularactin cytoskeleton in viable osteocytes in situ adheres to the principledirection of externalmechanical loading [64]. This indicates that indeedosteocyte cell bodies might be able to sense the external mechanicalloads and hence orientate in accordance with these loads.

In mammalian cells local physical forces are conveyed to the cell bymechanically coupling the cellular cytoskeletal network to the extracel-lular matrix via focal adhesions [65]. The focal adhesions are comprisedofmultiple actin-associated proteins such as paxillin, vinculin, talin, andzyxin [66,67]. A major constituent in focal adhesions, mediatingdownstream intracellular signaling is focal adhesion kinase (FAK).Focal adhesions are known to be involved in mechanosensation anddownstream signaling in various cell types, and external mechanicalforces have a direct role in their formation [65]. Paxillin proteins are pre-dominantly “localized” to upper and lower “poles” of fibular osteocytecell bodies, whereas they are evenly distributed across the osteocytecell bodies in calvaria suggesting that focal adhesions are formed inosteocytes along the direction of principle strains within the bone[64]. FAK is essential for mechanotransduction in osteoblasts [68], andFAK has a similar role in osteocyte mechanotransduction [69]. It wasfound that mechanical stimulation bymeans of a pulsatile fluid flow in-duced stabilization of β-catenin in osteocytes in a FAK-dependentmechanism [69]. Interestingly, knockdown of membrane-type matrixmetalloproteinase-1 (MT1-MMP) increased the number and size offocal adhesions in cultured MLO-Y4 osteocytes concomitantly with anenhanced NO production and c-jun and c-fos mRNA expression in re-sponse to mechanical stimulation [70]. This indicates that MT1-MMPknockdown osteocytes have an increased sensitivity to mechanicalloading and demonstrates a novel and unexpected potential role forMT1-MMP in mechanosensing.

Primary cilia are single cytoplasmic organelles found in virtually alleukaryotic cells. They protrude into the extracellular space from thecell surface and function as mechanosensors in tissues such as kidney.Osteocytes also possess a single primary cilium [71]. PKD1/PC1, amechanosensory protein in the kidney that localizes to primary cilia,is known to play a role in normal bone structure. It is not yet establishedif PKD1 functions via the primary cilia or it has a function in anotherlocation in the cell. Interestingly, MC3T3-E1 osteoblasts and MLO-Y4osteocytes possess primary cilia that project from the cell surfaceand deflect during fluid flow [72]. These primary cilia are requiredfor the osteocyte response to dynamic fluid flow in vitro. However,the location of the primary cilium, i.e. on the osteocyte cell body,makes it difficult to envision a role for the primary cilium as a flowsensor for osteocytes in vivo, because physical laws dictate thatloading-induced fluid flowwill primarily occur around the osteocytecell processes and it is difficult to envision how a primary cilia couldfit into the lacuno-canalicular space without being already severelybent [58,36]. An alternative hypothesis, postulated by Bell, suggeststhat cells sense hydraulic pressure by using the primary cilium as asensor of hydrostatic pressure, but no experimental evidence to supportthis hypothesis currently exists [73]. It remains a matter of debatewhether organelles situated on the cell body will contribute tomechanosensing in vivo, or that any mechanotransduction complexesare more likely to be located on the osteocyte processes.

It has been suggested that the osteocyte processes might be attacheddirectly to the canalicular wall by β3 integrins at the apex of infrequent,previously unrecognized canalicular projections [43]. A theoreticalmodel was developed that predicts that the tensile forces acting onthese integrins can be as large as 15 pN, and thus provide stable attach-ment in the range of physiological loading [20]. The model also predictsthat axial strains caused by the sliding of actinmicrofilaments relative tothe fixed attachments are two orders of magnitude greater thanwhole-tissue strains thereby producing local membrane strains in thecell process that can exceed 5%. In vitro experiments indicated thatmem-brane strains of this order are large enough to open stretch-activated

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cation channels [74]. It is likely that stretch-activated ion channels play arole in the transduction ofmechanical stimuli into a chemical response inosteocytes. A well known early response to mechanical stimulation ofosteocytes and other mechanosensitive bone cells in vitro is an increasein intracellular calcium concentration [75], which could well be causedby opening of stretch-activated ion channels. Although no directevidence exists that transient receptor potential (TRP) channels arestretch-activated ion channels, it is an idea that has often been put for-ward. In MLO-Y4 osteocytes the calcium response to mechanical stimu-lation can be partially blocked by Gd3+ [76], suggesting that some kindof TRP channel is involved in this response. Little is known about TRPchannel expression in osteocytes, only TRPV6 is known to be expressedat low levels inmurine osteocytes [77]. So far the involvement of specificion channels in the mechanoresponse of osteocytes has not beenelucidated.

As described above, actin microfilaments in the osteocyte cell exten-sions may slide relative to the fixed attachments. As a result, stretch-activated ion channels may be pulled open, since such channels areconnected to the cytoskeleton. On the outside of the osteocyte processany stretch-activated ion channelsmay also be connected to the extracel-lular matrix via tethers, further enabling the opening of ion channels.Such tethering filaments appear to be absent in the pericellular spacesurrounding the cell body, likely due to the wide pericellular space(~1 μm) between the cell membrane and the wall of the lacuna. In con-trast, the pericellular space surrounding the cell process on average is80 nm [32]. You et al. [78]were the first to propose that therewere reg-ularly spaced tethering filaments that attached the cell processes to thecanalicular wall. They postulated that the flow through the pericellularmatrix, which was supported by these tethers, would put them intension creating a hoop strain on the cell processmembrane. This hypo-thetical model predicted that membrane strains up to 100-fold largerthanwhole tissue strains could be generated by thismechanism. Subse-quently, You et al. [32] using refined fixation techniques demonstratedthe existence of these tethering filaments and also the organization ofthe central actin filament within the process. Immunostaining studiesdemonstrate the existence of CD44 [79] and αvβ3 integrin [43] in thematrix surrounding the process, suggesting that potentially CD44serves as the tethering molecule since it has an attachment site forhyaluronan. Interestingly, a protein tether involved in transduction ofmechanical stimuli has recently been identified in cutaneousmechano-receptors [80]. This molecule is a protein filament with a length of~100 nm. A major objection to the hoop strain, tether and integrintheories is that they are based on the impression that the dendriticprocesses are somewhat permanently anchored to the lacunar wall.However, osteocyte dendritic processes extend and retract over time, re-vealing that the osteocyte is highly dynamic [81]. Retraction would bedifficult to occur unless gap junctions at the apical end of the dendriticprocesseswere disrupted, but this could occur during naturally occurringapoptosis.

Connexins are essential for the communication of cells amongthemselves and with their environment. Considering that osteocytesform a vast interconnected network of cells that is much dependenton cell–cell connections for rapid transmission of signals, it is notsurprising that connexins play an important role in osteocyte func-tion. Specifically connexin 43 (Cx43) is essential for osteocytes, andmice lacking Cx43 in osteocytes exhibit increased osteocyte apopto-sis and empty lacunae in cortical bone [82]. In addition, osteoblastand osteocyte-specific Cx43-deficient mice displayed bone loss asa result of increased bone resorption and osteoclastogenesis [23].Although Cx43 seems to be an important mediator of mechanicalresponses of osteocytes in vitro [83,84] Cx43 deficient micedisplayed an enhanced anabolic response to mechanical load ratherthan a reduced response [23].

From these findings one may conclude that despite the longstanding recognition of the importance of mechanical loading formaintenance and adaptation of bone mass and structure, it is still a

mystery which (ultra)structural features are responsible for transducingloading-derived fluid flows into a signal that activates the osteocytes.

What constitutes the osteocyte response to mechanical stimulation?

Following mechanosensation and conversion of the mechanical sig-nal into a chemical signal, osteocytes orchestrate the formation and/oractivity of the osteoblasts and osteoclasts Fig. 4. The intercellular com-munication required for such a feat is achieved by the production of arange of biomolecules like nitric oxide (NO), prostaglandins, bonemor-phogenetic proteins,Wnts, andmany others (Fig. 5). Asmentioned pre-viously, an important early response tomechanical loading is the influxof calcium ions through ion channels in the plasma membrane and therelease of calcium from internal stores [29,30,44]. The rise in intracellu-lar calcium concentration activates many downstream signaling cas-cades such as protein kinase C and phospholipase A2, and is necessaryfor activation of calcium/calmodulin dependent proteins, such as theconstitutive forms of nitric oxide synthase (NOS). The activation ofphospholipase A2 results, among others, in the activation of arachidonicacid production and prostaglandin E2 (PGE2) release [85]. Other geneswhose expression in osteocytes is modified by mechanical loading in-clude c-fos, MEPE, and IGF-I [86].

NO is produced when L-arginine is converted to L-citruline in thepresence of NOS enzyme, molecular oxygen, NADPH, and other cofactors[87,88]. A wide range of studies have clearly demonstrated that me-chanical stimulation, both via directmanipulation of cells and via appli-cation of a fluid flow to cultured osteocytes, results in NO production[60,89–91]. NO has been shown to modulate the activity of osteoblastsand osteoclasts [15,16] and inhibition of NO production inhibited me-chanically induced bone formation in rats [92,93]. In contrast to popularbelief, it was recently found that expression of endothelial NOS (eNOS)protein is not necessary for mechanical stimulation-induced NO produc-tion by cultured osteoblasts [94]. We have confirmed that eNOS mRNAexpression is not detectable in MLO-Y4 osteocyte-like cells, which none-theless show a robust NO response to mechanical stimulation in vitro(unpublished observations). With the current interest in NO as anabolicagent for bone it is of interest to delineate which enzyme(s) is/areresponsible for NO production by mechanically stimulated osteocytes.

Prostaglandins are abundantly produced by osteocytes, as well asby other cells of the osteoblastic lineage [95–98], and play a keyrole in the bone formation response to mechanical loading in vivo[15,99]. Several studies have shown that osteocytes rapidly increasetheir prostaglandin production in response to mechanical loading invitro [99,100]. Cyclooxygenase (COX) is the key enzyme involved inthe production of prostaglandins [67], and exists in a constitutive(COX-1) and an inducible form (COX-2). Fluid shear stress does notaffect COX-1 mRNA expression in primary human bone cells [101],but mechanical loading induces a rapid rise in COX-2 mRNA inhuman bone cells and chicken osteocytes in vitro, as well as COX-2protein expression in rat bone cells in vivo [101–103]. Importantly, inhi-bition of COX-2, but not COX-1, inhibits fluid flow-induced prostaglandinproduction by primary bone cells in vitro [104]. In addition, COX-2 hasbeen shown to mediate the anabolic response of bone tissue to mechan-ical loading, illustrating the importance of loading-induced prostaglandinproduction for the process of adaptive bone remodeling. Prostaglandinsare released throughhemi-channels and purinergic receptors in responseto mechanical stimuli [105].

TheWnt family of proteins has been recently added to the repertoireof mediators of mechanotransduction in bone. Wnt signaling might bean important modulator of the process of mechano-regulated bone ad-aptation. Wnt signaling can be mediated by the β-catenin pathways,through kinases or through activation of GTPases, thereby modulatingcytoskeletal organization [106,107]. Activation of β-catenin signalingin response to fluid shear stress is likely mediated by PGE2 inMLO-Y4 osteocytes [108]. In light of the role of the cytoskeleton inmechanosensing, it is noteworthy thatWntsmaymodulate cytoskeletal

Fig. 4. Schematic overview of the role of osteocytes in the process of bone remodelling.

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organization, and thatβ-catenin links cadherins to the actin cytoskeleton.In vitro studies have shown that MC3T3-E1 osteoblasts increase Wntgene expression after mechanical stimulation by substrate deformation[47], and that pulsating fluid flow up-regulates mRNA expression ofβ-catenin, APC, and Wnt3a, as well as the Wnt antagonist SFRP4 inMLO-Y4 osteocytes [46], showing that osteocytes respond to mechanicalloadingwith amodulation of expression ofmolecules involved in thewntsinalling cascade. Recently it was shown that LRP5, a co-receptor forWntsignaling, functions locally in osteocytes. Mice with osteocyte-specificexpression of inducible Lrp5 mutations had bone properties comparableto those inmicewith inheritedmutations, demonstrating the importanceof wnt signalling for osteocytes [109].

Sclerostin appears to be highly expressed in mature osteocytescompared to immature osteocytes [48]. Sclerostin protein may betransported through canaliculi to the bone surface, where it inhibitsbone formation by osteoblasts. Studies in sclerostin-deficient transgenicmice suggest that sclerostin inhibits bonemass accrual. Themice lackingsclerostin exhibit an increased bone mass resembling the human condi-tion of sclerosteosis, which is due to a premature termination of the Sostgene [110] that transcribes sclerostin. Sclerostin acts as aWnt antagonistby binding the Wnt co-receptor Lrp5 [111], Lrp5 being an importantanabolic regulator of bone mass [109,112]. Interestingly, Sost tran-scripts and sclerostin protein levels were dramatically reduced in oste-ocytes after loading of mouse ulnae in vivo. The magnitude of the strainstimulus was associated with Sost staining intensity and number ofsclerostin-positive osteocytes. Hindlimb unloading on the other handyielded a significant increase in Sost expression in themouse tibia [113].

Othermolecules have been identifiedwhose expression ismodulatedby mechanical loading and seem to be more or less osteocyte-specific.MEPE is highly expressed in osteocytes as compared to osteoblasts.

Fig. 5. Schematic representation of some of the bio-molecules involved in chemi

MEPE plays an inhibitory role in bone formation in mice [114].MEPE-null mice show increased bone formation and bone mass as wellas resistance to age-related trabecular bone loss [114], suggesting a rolefor MEPE in the regulation of bone homeostasis. It has been shown thatMEPE expression is upregulated in a time-dependent fashion in alveolarosteocytes in response to mechanical loading applied by orthodontictooth movement [115], and MEPE expression is enhanced in osteocytessubjected to mechanical loading in vitro [116].

Dentin matrix protein 1 (DMP1) is another molecule that seems tobe highly expressed in osteocytes compared to other cells types[117,118]. A potential role of DMP1 in osteocytes may be related tohydroxyapatite formation. DMP1 is specifically expressed along andin the canaliculi of osteocytes within the bone matrix [117]. The can-aliculi and lacunae in bones of DMP1-null mice have a compromisedstructure, which can have implications for the amplification of loadsignals to the osteocytes [119]. DMP1 expression increases 2 to 3-foldin osteocytes of the mouse ulna at 24 h after a single 2.4 N load for30 s at 2 Hz [120]. Phex gene expression is also increased in responseto mechanical loading [120]. The precise function of Phex is unclearbut it clearly plays a role in phosphate homeostasis and bonemineralization.

Signaling molecules produced by mechanically-loaded osteocytesmodulate the recruitment and activity of osteoblasts and/or osteoclasts.Osteoblast recruitment and activity can be stimulated byprostaglandinsandWnts [121–123]. Fluidflow-subjected osteocytes stimulate alkalinephosphatase activity in osteoblasts [124]. NO also has an anabolic effecton osteoblast activity. Osteoclast activity seems to be inhibited by NOproduced in osteocytes [49]. MLO-Y4 osteocytes also produce M-CSF,RANKL, and OPG, and are thereby able to actively promote osteoclastformation and activity under static culture conditions. The promotion

cal signaling in bone and their effects on bone formation and/or resorption.

188 J. Klein-Nulend et al. / Bone 54 (2013) 182–190

of osteoclast formation depends on cell–cell contact, possibly due to therequirement of cell-bound RANKL [125,126]. Indeed it has been shownrecently that bone mass in adult mice is determined by RANKL producedby osteocytes rather than osteoblasts [15,16].

Conclusion

In bone, skeletal homeostasis is achieved by local osteoclast-mediateddegradation of the bone matrix and osteoblast-mediated formation ofnew bone matrix without compromising the overall architecture andanatomy of bone. This is achieved in accordance with the external me-chanical loading conditions to which the bone is subjected. Osteocytesplay a central role in this remodeling process by sensing the external me-chanical loads and then transmitting the information to the effector cells,the osteoblasts and/or the osteoclasts, which then maintain the skeletalhomeostasis.

Conflict of interest

All authors have no conflicts of interest.

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