The Osteocyte: from “Prisoner” to “Orchestrator”

Journal of Functional Morphology and Kinesiology

Review The Osteocyte: From “Prisoner” to “Orchestrator”

Carla Palumbo * and Marzia Ferretti

Section of Human Morphology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, 41124 Modena, Italy; [email protected] * Correspondence: [email protected]

Abstract: Osteocytes are the most abundant bone cells, entrapped inside the mineralized bone matrix. They derive from osteoblasts through a complex series of morpho-functional modiﬁcations; such modiﬁcations not only concern the cell shape (from prismatic to dendritic) and location (along the vascular bone surfaces or enclosed inside the lacuno-canalicular cavities, respectively) but also their role in bone processes (secretion/mineralization of preosseous matrix and/or regulation of bone remodeling). Osteocytes are connected with each other by means of different types of junctions, among which the gap junctions enable osteocytes inside the matrix to act in a neuronal-like manner, as a functional syncytium together with the cells placed on the vascular bone surfaces (osteoblasts or bone lining cells), the stromal cells and the endothelial cells, i.e., the bone basic cellular system (BBCS). Within the BBCS, osteocytes can communicate in two ways: by means of volume transmission and wiring transmission, depending on the type of signals (metabolic or mechanical, respectively) received and/or to be forwarded. The capability of osteocytes in maintaining skeletal and mineral homeostasis is due to the fact that it acts as a mechano-sensor, able to transduce mechanical strains into biological signals and to trigger/modulate the bone remodeling, also because of the relevant role of sclerostin secreted by osteocytes, thus regulating different bone cell signaling pathways. The authors want to emphasize that the present review is centered on the morphological aspects of the osteocytes that clearly explain their functional implications and their role as bone orchestrators. Citation: Palumbo, C.; Ferretti, M. The Osteocyte: From “Prisoner” to Keywords: osteocytes; bone mechano-sensor; skeletal homeostasis; mineral homeostasis; bone “Orchestrator”. J. Funct. Morphol. remodeling Kinesiol. 2021, 6, 28. https://doi.org/ 10.3390/jfmk6010028

Academic Editor: Pietro Scicchitano 1. Osteoblast-to-Osteocyte Transformation It has been known for more than a century [1–3] that the osteocyte originates from the Received: 5 February 2021 osteoblast. However, the process of osteoblast-to-osteocyte differentiation has been widely Accepted: 11 March 2021 Published: 17 March 2021 investigated only at a later time point with regard to both morphological and functional aspects. The structural differences between osteoblasts and osteocytes were shown by vari-

Publisher’s Note: MDPI stays neutral ous authors in the 1960s to1980s [4–8], but only afterwards was established the temporal with regard to jurisdictional claims in sequence of the events that allows the transformation of the prismatic osteoblast (generally published maps and institutional affil- arranged in laminae facing the vascular bone surfaces) into the dendritic mature osteocyte iations. (embedded in the mineralized matrix) [9–11]. Concerning the dynamic modification of the cell body of preosteocyte (i.e., the differentiating osteocyte), the amount of the cytoplasmic organelles decreases, whereas the nucleus-to-cytoplasm ratio increases depending on the diminution of its secretive activity [9]. In parallel to both the cellular body reduction in size and the modification in ultrastructure, the formation of the cytoplasmic processes proceeds Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. with an asynchronous and asymmetric pattern, considering that the cells in differentiation This article is an open access article are progressively further away from the vascular surface due to the osteoid secreted by distributed under the terms and the osteoblastic lamina (Figure1): firstly, the differentiating osteocytes maintain contacts conditions of the Creative Commons with the mature osteocytes that recruited them from the osteoblastic lamina, forming short Attribution (CC BY) license (https:// “mineral” processes; later, they establish contacts with the migrating osteoblastic lamina, creativecommons.org/licenses/by/ elongating slender and long “vascular” processes, issued before the complete mineraliza- 4.0/). tion of the surrounding osteoid. The asynchronous and asymmetrical dendrogenesis is the

J. Funct. Morphol. Kinesiol. 2021, 6, 28. https://doi.org/10.3390/jfmk6010028 https://www.mdpi.com/journal/jfmk J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 2 of 18

J. Funct. Morphol. Kinesiol. 2021, 6, 28 2 of 17 with the migrating osteoblastic lamina, elongating slender and long “vascular” processes, issued before the complete mineralization of the surrounding osteoid. The asynchronous and asymmetrical dendrogenesis is the expression of the fact that osteocytes (as all bone expressioncells) live in of an the asymmetrical fact that osteocytes environment, (as all bone between cells) live the in mature an asymmetrical mineralized environment, matrix and betweenthe vascular the maturesurface mineralized(covered by osteoblastic matrix and thelaminae vascular or bone surface lining (covered cells); thus, by osteoblastic it is logical laminaeto expect or that bone not lining only cells);the osteocytes, thus, it is but logical also to the expect preosteocytes that not only and thethe osteocytes,osteoblasts, but are alsomorpho the preosteocytes‐functionally asymmetric and the osteoblasts, cells. are morpho-functionally asymmetric cells.

FigureFigure 1.1. Schematic drawing drawing showing showing the the asynchronous asynchronous and and asymmetric asymmetric pattern pattern of ofcytoplasmic cytoplasmic processes formation during osteocyte differentiation (preosteocytes = black; osteoblasts = white). processes formation during osteocyte differentiation (preosteocytes = black; osteoblasts = white). (A) Preosteocyte enlarges its secretory territory, thus reducing its appositional growth rate, and (A) Preosteocyte enlarges its secretory territory, thus reducing its appositional growth rate, and starts to radiate processes towards the osteoid. (B) Preosteocyte, located inside the osteoid seam startsbut still to in radiate contact processes with the towards osteoblastic the osteoid.lamina, continues (B) Preosteocyte, to radiate located short and inside thick the cytoplasmic osteoid seam butprocesses still in only contact from with its mineral the osteoblastic‐facing side. lamina, (C) Preosteocyte, continues to before radiate being short completely and thick cytoplasmicburied by processesminerals, radiates only from long its and mineral-facing slender processes side. ( Cfrom) Preosteocyte, its vascular‐ beforefacing beingside to completely remain in touch buried with by minerals,the osteoblastic radiates lamina. long and slender processes from its vascular-facing side to remain in touch with the osteoblastic lamina. At the end of the process, the osteocytes are confined to lacuno‐canalicular cavities, “prisoners”At the end inside of thethe process,mineralized the osteocytesmatrix. Despite are confined this fact, to they lacuno-canalicular are connected, thanks cavities, to “prisoners”the dendrogenesis inside the process, mineralized to each matrix. other Despiteand with this the fact, bone they cells are on connected, the vascular thanks surfaces to the dendrogenesisthrough a network process, of todendrities, each other running and with within the bone the cells canalicular on the vascular network; surfaces this condition through aallows network osteocytes of dendrities, to act as running “orchestrators” within the of bone canalicular processes network; [12,13]. this Prerequisite condition for allows that osteocytesis the existence to act of as junctional “orchestrators” complexes of bone occurring processes among [12 ,osteocyte13]. Prerequisite cytoplasmic for that processes is the existence[7,14,15], of suggesting junctional complexesthat the bone occurring cells of among the osteogenic osteocyte cytoplasmiclineage, arranged processes in [network7,14,15], suggesting(Figure 2), might that the act bone as a cells functional of the osteogenicsyncytium, lineage, that includes arranged also in the network cells covering (Figure 2the), mightvascular act bone as a functional surfaces, bone syncytium, lining cells that includes[15] or osteoblasts also the cells [16]. covering the vascular bone surfaces, bone lining cells [15] or osteoblasts [16]. In conclusion, throughout the whole differentiation process, preosteocytes are always in close relationship with the neighboring cells (osteoblasts, osteocytes) by means of variously-shaped intercellular contacts (invaginated finger-like, side-to-side, and end to- end) and two types of specialized junctions: gap and adherens [14]. The pivotal role played by these contacts and junctions in osteocyte differentiation and activity will be discussed in the context of their distinct functional significance. J. Funct. Morphol. Kinesiol. 2021, 6, 28 3 of 17 J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 3 of 18

FigureFigure 2.2. Transmission electronelectron microscopemicroscope (TEM)(TEM) micrographmicrograph showing the continuous cytoplasmiccytoplas‐ mic network of the cells of the osteogenic lineage, extending from osteocytes to endothelial cells. network of the cells of the osteogenic lineage, extending from osteocytes to endothelial cells. (PO (PO preosteocyte, OB osteoblast, SC stromal cell, EC endothelial cell). ×22,500. preosteocyte, OB osteoblast, SC stromal cell, EC endothelial cell). ×22,500.

2. OsteogenesisIn conclusion, Processes throughout and Osteocytethe whole Morphology differentiation process, preosteocytes are al‐ ways in close relationship with the neighboring cells (osteoblasts, osteocytes) by means of To fully understand the osteocyte morphology in the different contexts in which it variously‐shaped intercellular contacts (invaginated finger‐like, side‐to‐side, and end to‐ originates, it is important to mention that morphological studies have shown that osteocyte end) and two types of specialized junctions: gap and adherens [14]. The pivotal role differentiation is quite different in two types of osteogenesis. We clearly demonstrated, played by these contacts and junctions in osteocyte differentiation and activity will be for the first time in 2002 in our labs, that not all osteoblasts are arranged in the well- discussed in the context of their distinct functional significance. known movable laminae: the two types of osteogenesis were named, during our studies on intramembranous ossification, “static bone formation” or static osteogenesis (SO) and 2. Osteogenesis Processes and Osteocyte Morphology “dynamic bone formation” or dynamic osteogenesis (DO) [17–21]. SO and DO occur in temporalTo fully sequence understand and depend the osteocyte on whether morphology or not osteoblast in the different movement contexts occurs. in We which pointed it outoriginates, that the it well-known is important dynamic to mention osteogenesis that morphological (DO), performed studies have by migrating shown that osteoblast osteo‐ laminae,cyte differentiation is preceded is byquite static different osteogenesis in two (SO)types in of which, osteogenesis. at the onsetWe clearly of ossification, demon‐ cordsstrated, of for stationary the first osteoblaststime in 2002 (instead in our labs, of laminae that not ofall movable osteoblasts osteoblasts) are arranged transform in the intowell osteocytes‐known movable in the samelaminae: site wherethe two they types differentiated of osteogenesis from were mesenchymal named, during progenitor our elements.studies on In intramembranous SO (Figure3A,B), ossification, stationary osteoblasts“static bone (acting formation” in sites or wherestatic osteogenesis no bone pre- exists)(SO) and are “dynamic in very peculiar bone formation” and unusual or conditions dynamic osteogenesis with respect to(DO) the [17–21]. well-known SO and dynamic DO osteoblasts,occur in temporal arranged sequence in laminae and depend and secreting on whether bone or closenot osteoblast to the preexistent movement one: occurs. i.e., theyWe pointed are in a out symmetrical that the well environment‐known dynamic since osteogenesis the osteoblast (DO), cords performed differentiate by migrating at about halfosteoblast distance laminae, between is preceded two vessels; by static in this osteogenesis extent, several (SO) in factors which, are at the involved, onset of such ossifi as‐ endothelial-cell-derivedcation, cords of stationary cytokines osteoblasts (Endothelin-1) (instead of and laminae growth of factorsmovable (EDGF, osteoblasts) PDGF). trans In fact,‐ theyform give into rise osteocytes to big globous in the same osteocytes, site where often they located differentiated inside confluent from mesenchymal lacunae, with pro short‐ andgenitor symmetrical elements. dendrites In SO (Figure that 3A,B), can radiate stationary simultaneously osteoblasts from(acting all in around sites where their cellno bone body becausepre‐exists) they are are in completely very peculiar surrounded and unusual by the conditions unmineralized with matrix.respect Into DOthe (Figurewell‐known3C,D), instead,dynamic osteoblasts osteoblasts, transform arranged intoin laminae small ovoidal/ellipsoidal and secreting bone close spidery to the osteocytes preexistent inside one: an asymmetricali.e., they are in environment, a symmetrical whose environment dendrites since form the inosteoblast an asynchronous cords differentiate and asymmetrical at about mannerhalf distance in concomitance between two with, vessels; and in depending this extent, on, several the advancing factors are mineralizing involved, such surface as en and‐ thedothelial migrating‐cell‐derived osteogenic cytokines laminae. (Endothelin‐1) and growth factors (EDGF, PDGF). In fact, they give rise to big globous osteocytes, often located inside confluent lacunae, with short J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 4 of 18

and symmetrical dendrites that can radiate simultaneously from all around their cell body because they are completely surrounded by the unmineralized matrix. In DO (Figure 3C,D), instead, osteoblasts transform into small ovoidal/ellipsoidal spidery osteocytes in‐ J. Funct. Morphol. Kinesiol. 2021, 6, 28 side an asymmetrical environment, whose dendrites form in an asynchronous and asym4 of 17‐ metrical manner in concomitance with, and depending on, the advancing mineralizing surface and the migrating osteogenic laminae.

Figure 3. Schematic drawing showing the occurrence, in sequence, of static (SO) and dynamic Figure 3. Schematic drawing showing the occurrence, in sequence, of static (SO) and dynamic (DO) (DO) osteogenesis. In SO, stationary osteoblast cords (A) differentiate at about half distance be‐ osteogenesis. In SO, stationary osteoblast cords (A) differentiate at about half distance between two tween two vessels, giving rise to big globous osteocytes (B), often located inside confluent lacunae, vessels,with short giving and rise symmetrical to big globous dendrites. osteocytes In DO, (B osteoblastic), often located laminae inside differentiate conﬂuent lacunae, on the surface with short of andSO‐derived symmetrical trabeculae dendrites. (C) and In DO, transform osteoblastic into small laminae spidery differentiate osteocytes on (D the) whose surface asymmetrical of SO-derived trabeculaedendrites form (C) and in concomitance transform into with small the spidery migration osteocytes of the osteogenic (D) whose lamina. asymmetrical See text dendrites for explana form‐ intion. concomitance (EDGF: endothelial with the‐derived migration growth of the factor; osteogenic PDGF: lamina.platelet‐derived See text growth for explanation. factor). (EDGF: endothelial-derived growth factor; PDGF: platelet-derived growth factor). Among all osteocytes (both SO‐ and DO‐derived) and between osteocytes and oste‐ oblasts,Among simple all contacts osteocytes and (both specialized SO- and gap DO-derived) junctions were and observed, between osteocytesthat allow andall bone osteoblasts,cells to remain simple functionally contacts and connected; specialized therefore, gap junctions a continuous were observed, osteocyte that network allow all extends bone cellsthroughout to remain the functionally bone, regardless connected; of its therefore,static or dynamic a continuous origin. osteocyte This cellular network network extends has throughoutthe characteristics the bone, of a regardless functional of syncytium, its static or potentially dynamic origin.capable This of modulating, cellular network by wiring has thetransmission characteristics (see ofbelow), a functional the cells syncytium, of the osteogenic potentially lineage capable covering of modulating, the bone by surfaces. wiring transmissionMorphology, (see as well below), as density the cells and of distribution the osteogenic of osteocytes, lineage covering is not only the bone related surfaces. to the Morphology,type of osteogenesis as well asfrom density which and they distribution derive but ofalso osteocytes, to the collagen is not onlytexture related where to they the typeare located, of osteogenesis on whose from spatial which organization they derive the but osteocytes also to the play collagen a pivotal texture role where[22]. Unlike they arewhat located, was traditionally on whose spatial reported organization in textbooks the in osteocytes relation to playbone ahistology, pivotal role about [22 3]. decades Unlike whatago Marotti was traditionally and collaborators reported reworked in textbooks the inclassification relation to boneof bone histology, tissues,about demonstrating, 3 decades ago Marotti and collaborators reworked the classification of bone tissues, demonstrating, by means of both SEM (scanning electron microscopy) and TEM (transmission electron by means of both SEM (scanning electron microscopy) and TEM (transmission electron microscopy) analyses, that woven bone can be arranged in two different tissues [10,22– microscopy) analyses, that woven bone can be arranged in two different tissues [10,22–24]: 24]: “not lamellar woven bone” mostly formed by SO and “lamellar woven bone” mostly “not lamellar woven bone” mostly formed by SO and “lamellar woven bone” mostly formed by DO. Comparing the two type of tissue (Figure 4): in not‐lamellar‐woven‐bone formed by DO. Comparing the two type of tissue (Figure4): in not-lamellar-woven-bone (Figure 4A), osteocytes are symmetric, randomly distributed in clusters, with a large and (Figure4A), osteocytes are symmetric, randomly distributed in clusters, with a large and irregularly globous shape of the cell body; in lamellar‐woven‐bone (Figure 4B) (made up irregularly globous shape of the cell body; in lamellar-woven-bone (Figure4B) (made of alternate dense and loose lamellae, both with an interwoven fibrous texture), osteocytes up of alternate dense and loose lamellae, both with an interwoven ﬁbrous texture), os- are asymmetric, distributed in planes exclusively located inside loose lamellae, with an teocytes are asymmetric, distributed in planes exclusively located inside loose lamellae, with an almond-like shape of the cell body (i.e., a triaxial ellipsoid) [10,25–28]. It is to be underlined that, generally, morphology of osteocytes is indirectly inferred from the shape of the lacuno-canalicular network in which they are enclosed rather than directly from their protoplasm. J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 5 of 18

almond‐like shape of the cell body (i.e., a triaxial ellipsoid) [10,25–28]. It is to be under‐ J. Funct. Morphol. Kinesiol. 2021, 6, 28 lined that, generally, morphology of osteocytes is indirectly inferred from the shape 5of of the 17 lacuno‐canalicular network in which they are enclosed rather than directly from their pro‐ toplasm.

FigureFigure 4. 4.( (AA,B, B):): Schematic Schematic drawing drawing showing showing arrangement arrangement of of osteocytes osteocytes and and the the surrounding surrounding colla- col‐ lagen fibers in not‐lamellar‐woven‐bone (A) and in lamellar‐woven‐bone (B). In both types of bone gen fibers in not-lamellar-woven-bone (A) and in lamellar-woven-bone (B). In both types of bone tissue, osteocyte lacunae (blue ovals) are surrounded by perilacunar matrices of loosely arranged tissue, osteocyte lacunae (blue ovals) are surrounded by perilacunar matrices of loosely arranged collagen fibers. In lamellar‐woven‐bone, the loose lamellae result as a consequence of alignment collagenand fusion fibers. of the In perilacunar lamellar-woven-bone, loose matrices the of loose the osteocytes lamellae result arranged as a in consequence planes; the ofdense alignment bun‐ anddles fusion of collagen of the fibers perilacunar do not loose contain matrices osteocytes. of the ( osteocytesC, D): SEM arranged micrographs in planes; of not the‐lamellar dense‐ bundleswoven‐ ofbone collagen (C) and fibers lamellar do not‐woven contain‐bone osteocytes. (D); the osteocyte (C,D): SEM lacunae micrographs are larger, of not-lamellar-woven-bonemore numerous, and ir‐ (Cregularly) and lamellar-woven-bone distributed in not‐lamellar (D); the‐woven osteocyte‐bone lacunae with respect are larger, to lamellar more numerous,‐woven‐bone, and irregularly where distributedthey are only in not-lamellar-woven-bonelocated inside loose lamellae. with Note respect that to dense lamellar-woven-bone, lamellae, alternating where with they the loose are only locatedones, are inside thinner loose and lamellae. do not contain Note that osteocyte dense lamellae, lacunae. alternating with the loose ones, are thinner and do not contain osteocyte lacunae. But we can ask: “was the egg or the hen born first?” that is to wonder if osteocyte affectsBut the we collagen can ask: texture “was or the vice egg‐versa or the the hen collagen born first?” texture that modifies is to wonder the cell morphology if osteocyte affectsand arrangement? the collagen textureThe fact or that vice-versa lamellar the bone collagen appears texture to be modifies a variety the of cellwoven morphology bone and andthat arrangement? osteocytes are The located fact that in loose lamellar lamellae bone appearsonly has to suggested be a variety that of the woven differences bone and in thattexture osteocytes between are lamellar located‐woven in loose and lamellae not‐lamellar only‐woven has suggested bone depend that theon the differences distribution in textureof osteocytes between throughout lamellar-woven the bone and matrix, not-lamellar-woven that is to say, boneon the depend manner on of the recruitment distribution of ofthe osteocytes osteocyte throughout‐differentiating the osteoblasts bone matrix, from that the is toosteogenic say, on the laminae manner [22,23,29]. of recruitment In not‐ ofla‐ themellar osteocyte-differentiating‐woven‐bone (generally osteoblasts laid down from very the rapidly) osteogenic the osteoblasts laminae [22 that,23 ,differentiate29]. In not- lamellar-woven-boneinto osteocytes are recruited (generally haphazardly laid down and very “enter” rapidly) the the bone osteoblasts in a random that differentiatefashion. As a intoresult, osteocytes such type are of recruited not‐lamellar haphazardly‐woven bone and consists “enter” of the an bone irregular in a random distribution fashion. of oste As‐ aocyte result,‐rich such areas type where of not-lamellar-woven the collagen is loosely bone arranged consists (since of an it corresponds irregular distribution to that of the of osteocyte-richperilacunar matrices) areas where (Figure the 4A,C). collagen is loosely arranged (since it corresponds to that of the perilacunarIn lamellar matrices)‐woven‐bone, (Figure whose4A,C). matrix is usually laid down at a lower rate than that of notIn‐lamellar lamellar-woven-bone,‐woven‐bone, the whose recruitment matrix isof usuallythe osteoblasts laid down that at differentiate a lower rate into than os‐ thatteocytes of not-lamellar-woven-bone, occurs in an orderly manner. the recruitment Since the cellular of the lamellae osteoblasts are that only differentiate those formed into by osteocytesloose texture, occurs this in suggests an orderly that manner. the osteoblasts Since the cellular committed lamellae to differentiating are only those formedinto osteo by‐ loosecytes texture, are recruited this suggests in successive that the groups osteoblasts and that committed those pertaining to differentiating to each into group osteocytes are dis‐ aretributed recruited in a in single successive plane, groups namely and that that corresponding those pertaining to a toloose each lamella. group are Thus, distributed loose la‐ inmellae a single could plane, simply namely form that as correspondinga result of the alignment to a loose lamella. and fusion Thus, of loosethe loosely lamellae‐arranged could simply form as a result of the alignment and fusion of the loosely-arranged fibers pertaining to the loose perilacunar matrices of the osteocytes they contain (Figure4B,D). Moreover, van Oers and coworkers [28] showed interactions between osteocyte shape and mechanical loading. In particular, their correlation seems to be related to the orientation of collagen fibers during osteoblast bone deposition: collagen gives bone its tensile strength J. Funct. Morphol. Kinesiol. 2021, 6, 28 6 of 17

which, in turn, conditions the elongation of osteocyte axes in relation to the direction of stress lines. These interactions are twofold: firstly, as regards the mechanical loading affecting the shape of osteocyte lacunae; secondly, as regards the shape of osteocytes influencing their mechano-sensibility and subsequent control of bone remodeling (see below). Shape variations of the lacuna hosting the osteocyte will also alter the direct cell strain, as well as the fluid flow and, in case, the microdamage inputs to the osteocyte. Furthermore, the shape of the osteocyte cell body affects its sensitivity to these inputs [30].

3. Osteocyte Network and Communications Morphological investigations [31,32] showed that an osteogenic cellular system is present inside the bone, formed by various cell types from depth to the vascular surfaces: osteocytes, osteoblasts, or bone lining cells (according to whether the bone surfaces are in growing or resting phase, respectively), stromal cells, and endothelial cells. They constitute a functional syncytium whose cells play different roles and have different relationships with the surrounding environment. Osteocytes are enclosed inside bone microcavities filled with the bone fluid compartment (BFC); the stromal cells, extending from vascular endothelia to the cells carpeting the bone surfaces, are bathed by the perivascular interstitial fluid (PIF); osteoblasts and/or bone lining cells, located at the interface “mineralized matrix/vascular surfaces”, separate the two different fluid compartments and are in contacts both with the “deep” and “superficial” cell populations. Intercellular contacts (simple contacts and/or specialized junctions) were observed throughout all cells of the osteogenic system [11,14]. Thus, these cells form a continuous protoplasmic network that extends from the osteocytes to the vascular endothelia [33]. The presence of gap junctions (allowing electric coupling) suggests that the bone osteogenic cells could be considered a functional syncytium regulated not only by diffusion through the intercellular fluids (volume transmission) but also by signals issued through the cytoplasmic network and driven through cytoplasmic extentions (wiring transmission) (see below). In the resting phase (namely, when no bone formation/erosion occur massively) osteocytes, bone lining cells, and stromal cells were named, as a whole, the bone basic cellular system (BBCS) (Figure5)[ 31,32], because they represent the only permanent cellular background capable of perceiving mechanical strains and biochemical factors and then of triggering and driving both processes of bone formation and bone resorption. It is appropriate to underline, in this context, that the majority of researchers consider osteoblasts and osteoclasts as the only relevant bony cells. Here, the authors want to emphasize that such a view is incorrect and misleading: indeed, osteoblasts and osteoclasts (the bone forming and bone reabsorbing cells, respectively) are transient cells, thus they cannot be the first to be involved, in the resting phase, in sensing skeletal requirements which modulate bone processes. Briefly, according to our view, osteoblasts and osteoclasts represent the “arms” of a worker; the actual “operation center” (i.e., the “brain”) is con- stituted by the cells of the osteogenic lineage in the resting phase (BBCS), particularly the osteocytes. (Figure6)[ 34]. As shown by our previous studies, signaling throughout BBCS can occur by volume transmission (VT) and/or wiring transmission (WT) [31,32,35,36]. VT corresponds to the routes followed by soluble substances (hormones, cytokines, growth factors), whereas WT represents the diffusion of ionic currents along cytoplasmic processes in a neuron-like manner. It is likely that biochemical signals first affect stromal cells embedded in PIF and diffuse by VT to reach the other cells of BBCS, whereas mechanical agents are firstly sensed by osteocytes bathed by BFC inside the mineralized matrix and then issued throughout BBCS by WT (Figure7). J. Funct. Morphol. Kinesiol. 2021, 6, 28 7 of 17 J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 7 of 18

J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 7 of 18

FigureFigure 5. 5.Schematic Schematic drawing drawing of of the the cells cells of of the the osteogenic osteogenic lineage lineage in in the the resting resting phase, phase, the the Bone Bone Basic Basic Cellular System. From left to right: osteocytes (OC), bone lining cells (BLC), stromal cells CellularFigure 5. System. Schematic From drawing left to right:of the osteocytes cells of the (OC), osteogenic bone lininglineage cells in the (BLC), resting stromal phase, cells the (SC) Bone and (SC)Basic and Cellular one vascular System. capillary.From left Thisto right: cell osteocytesnetwork forms (OC), a bonefunctional lining syncytium cells (BLC), since stromal cells cells are one vascular capillary. This cell network forms a functional syncytium since cells are joined by gap joined(SC) and by onegap vascular junctions capillary. (red circles). This cell network forms a functional syncytium since cells are junctions (red circles). joined by gap junctions (red circles).

Figure 6. Schematic drawing showing the percentage of the various bone cells present in the bone restingFigure 6.phase: Schematic 94% BBCS, drawing 5% showingosteoblasts, the and percentage 1% osteoclasts of the various (data from bone Parfitt cells present[34]). See in text the forbone Figure 6. Schematic drawing showing the percentage of the various bone cells present in the bone explanation.resting phase: 94% BBCS, 5% osteoblasts, and 1% osteoclasts (data from Parfitt [34]). See text for resting phase: 94% BBCS, 5% osteoblasts, and 1% osteoclasts (data from Parﬁtt [34]). See text explanation. for explanation. J. Funct. Morphol. Kinesiol. 2021, 6, 28 8 of 17 J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 8 of 18

Figure 7. Schematic picture of the BBCS, in which signal transmissions can occur by volume trans‐ Figure 7. Schematic picture of the BBCS, in which signal transmissions can occur by volume transmission (VT) and/or wiring transmission (WT): (A) VT corresponds to the routes followed by solu‐ mission (VT) and/or wiring transmission (WT): (A) VT corresponds to the routes followed by soluble ble substances; (B) WT represents the diffusion of ionic currents along cytoplasmic processes. Bio‐ substances;chemical signals (B) WT (hormones represents diffusing the diffusion from of the ionic blood, currents cytokines, along etc.) cytoplasmic first affect processes. stromal cells Biochemi- and caldiffuse signals by (hormonesVT to reach diffusing the other from cells theof BBCS, blood, whereas cytokines, mechanical etc.) first agents affect stromal are firstly cells sensed and diffuse by byosteocytes VT to reach and the then other issued cells throughout of BBCS, whereas BBCS by mechanical WT. agents are firstly sensed by osteocytes and then issued throughout BBCS by WT. It should be noted that besides WT, other similarities do exist between osteocytes and neurons.It should The short be noted and thatthick besides mineral WT, cytoplasmic other similarities processes do of exist osteocytes between resemble osteocytes neu‐ andronal neurons. dendrites, The whereas short and the thick long mineraland slender cytoplasmic vascular processescytoplasmic of osteocytesprocesses are resemble similar neuronalto neuronal dendrites, axons. Transmission whereas the of long signals and through slender osteocytes vascular cytoplasmicseems to occur processes by gap junc are‐ similartions instead to neuronal of synapses, axons. though Transmission it has been of signalsrecently through shown that osteocytes osteocytes seems produce to occur typ‐ byical gap neurotransmitters, junctions instead such of synapses, as nitric thoughoxide [37–40] it has beenand prostaglandin recently shown E2 that [38,41]. osteocytes Addi‐ produce typical neurotransmitters, such as nitric oxide [37–40] and prostaglandin E2 [38,41]. tionally, about two decades ago we provided evidence that WT occurs along osteocytes Additionally, about two decades ago we provided evidence that WT occurs along osteocytes in amphibian and murine cortical bone depending on loading [42,43], demonstrating that in amphibian and murine cortical bone depending on loading [42,43], demonstrating that osteocytes and bone lining cells are at the origin of ionic currents, by operating as a cellular osteocytes and bone lining cells are at the origin of ionic currents, by operating as a cellular membrane partition which regulates the ionic flows between bone (BFC) and plasma membrane partition which regulates the ionic flows between bone (BFC) and plasma (PIF). (PIF). Such osteocyte ionic currents are constantly directed to the bone lining cells and Such osteocyte ionic currents are constantly directed to the bone lining cells and stromal stromal cells during the resting phase. cells during the resting phase. As regards the transmission of mechanical signals, both recent and less recent litera‐ As regards the transmission of mechanical signals, both recent and less recent lit- ture indicates the osteocyte as the main strain‐sensitive bone cell [44–58]. We have shown erature indicates the osteocyte as the main strain-sensitive bone cell [44–58]. We have [42,43] that shear stress‐activated osteocytes are capable of steadily increasing and main‐ shown [42,43] that shear stress-activated osteocytes are capable of steadily increasing and taining the basal current produced by the ionic fluxes (streaming potentials), which occur maintaining the basal current produced by the ionic fluxes (streaming potentials), which occurinside inside the lacuno the lacuno-canalicular‐canalicular microcavities microcavities in inresponse response to to pulsing pulsing mechanical loadingloading (Figure(Figure8 ).8). Briefly,Briefly, thethe factfact that all osteocytes take take part part in in the the formation formation of of a apotential potential os‐ osteocyteteocyte syncytium syncytium supports supports the the view view that that mechanical mechanical signals throughout bonebone cellscells are are mainlymainly issuedissued byby wiringwiring transmissiontransmission sincesince volumevolume transmissiontransmission doesdoes notnot needneed cellcell con-con‐ tactstacts in in order order to to occur. occur. A A functional functional syncytium syncytium as as such such is is able able to to initiate, initiate, perform perform and and stop stop bonebone remodelingremodeling inin orderorder to to meet meet current current metabolic metabolic andand mechanical mechanical bonebone requirements.requirements. InIn thisthis context, the the osteocyte osteocyte role, role, played played in in bone bone adaptation adaptation to mechanical to mechanical stimuli, stimuli, is well is wellexplained explained by the by “Mechanostat the “Mechanostat Setpoint Setpoint Theory” Theory” (Figure (Figure 9), according9), according to which to which the oste the ‐ osteocytesocytes are arelikely likely capable capable of ofmodulating modulating bone bone resorption resorption and and bone bone formation formation within within a arange range of of physiological physiological mechanical mechanical strains strains whose whose upper upper and and lower lower limits limits are arenamed named set‐ points. When the lower (about 50 μ∑) and upper (about 2500 μ∑) setpoint values are ex‐ ceeded bone resorption and bone formation, respectively, are triggered. [44,59–61]. J. Funct. Morphol. Kinesiol. 2021, 6, 28 9 of 17

J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 9 of 18

setpoints. When the lower (about 50 µ∑) and upper (about 2500 µ∑) setpoint values are exceeded bone resorption and bone formation, respectively, are triggered [44,59–61].

J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 9 of 18

Figure 8. SEM micrograph of a portion of an osteon (A) showing the lacuno-canalicular cavities, on which the bone cellular Figure 8. SEM micrograph of a portion of an osteon (A) showing the lacuno‐canalicular cavities, on which the bone cellular systemFigure has 8. SEM been micrograph superimposed of a (in portion (B)), toof explainan osteon the (A events) showing occurring the lacuno during‐canalicular mechanical cavities, stress: inon response which the to bone mechanical cellular system has been superimposed (in B), to explain the events occurring during mechanical stress: in response to mechanical loading,system has bone been micro-deformations superimposed (in (induced B), to explain by loading the events on bone) occurring produce during ionic mechanical ﬂuxes (streaming stress: in potentials)response to in mechanical the bone loading, bone micro‐deformations (induced by loading on bone) produce ionic fluxes (streaming potentials) in the bone loading, boneﬂuid compartmentmicro‐deformations that, in turn, (induced activate the by osteocytes loading viaon shear bone) stress. produce Osteocytes, ionic by fluxes means (streaming of wiring transmission, potentials) issue in the bone fluid compartment that, in turn, activate the osteocytes via shear stress. Osteocytes, by means of wiring transmission, issue fluid compartmentsignals to the that, cells in covering turn, activate the bone surface,the osteocytes thus activating via shear osteoblast stress. recruitment Osteocytes, and bone by means formation. of wiring Vessel (red transmission, cylinder issue signals to thesignals cells to coveringthe cells covering the bone the bonesurface, surface, thus thus activating activating osteoblast osteoblast recruitment recruitment and andbone bone formation. formation. Vessel (red Vessel cyl‐ (red cyl‐ insideinder theinside Haversian the Haversian canal); canal); stromal stromal cell (light cell blue); (light bone blue); lining bone cell lining (green); cell (green); osteocyte osteocyte (yellow). (yellow). inder inside the Haversian canal); stromal cell (light blue); bone lining cell (green); osteocyte (yellow).

FigureFigure 9. 9. Schematic illustration of of Frost’s Frost’s Mechanostat Mechanostat Setpoint Setpoint Theory. Theory. The The setpoint setpoint values values are are expressed in microstrains (μ∑) and the bone mechanoreceptors (osteocytes) are activated only expressed in microstrains (µ∑) and the bone mechanoreceptors (osteocytes) are activated only when when the physiological setpoints are exceeded. In the “disuse” window, bone is lost owing to in‐ the physiological setpoints are exceeded. In the “disuse” window, bone is lost owing to increased creased resorption. In the “overuse” window, bone is gained owing to increased bone formation. resorption. In the “overuse” window, bone is gained owing to increased bone formation. Figure 9. Schematic illustration of Frost’s Mechanostat Setpoint Theory. The setpoint values are 4.4. OsteocytesOsteocytes asas BoneBone MechanicalMechanical Sensors Sensors and and Transducers Transducers of of Mechanical Mechanical Strains Strains into into expressed in microstrains (μ∑) and the bone mechanoreceptors (osteocytes) are activated only BiologicalBiological Signals Signals when the physiological setpoints are exceeded. In the “disuse” window, bone is lost owing to in‐ LozuponeLozupone andand coworkerscoworkers [[62]62] showedshowed inin vitrovitro thatthat thethe numbernumber ofof gapgap junctionsjunctions be-be‐ creased resorption. In the “overuse” window, bone is gained owing to increased bone formation. tweentween osteocytesosteocytes increasesincreases inin answeranswer toto thethe mechanical mechanical load load applied applied to to bone bone segments segments placedplaced inin cultures.cultures. This This is is in in line line with with the the suggestion suggestion that that gap gap junctions junctions play play an animportant impor- 4. Osteocytestantrole roleboth both inas cell Bone in‐ cell-to-cellto‐cell Mechanical communications communications Sensors and andin and cell in Transducerssynchronization, cell synchronization, of enabling Mechanical enabling small small mol Strains‐ into Biologicalmoleculesecules Signalsto be to beexchanged exchanged between between the the coupled coupled cells cells [11,14–16,63]; [11,14–16,63]; it it is is also also in lineline withwith the the fact that osteocytes are considered as mechanoreceptors, able to give rise to the transduction Lozuponefact that osteocytes and coworkers are considered [62] as showed mechanoreceptors, in vitro that able theto give number rise to theof gaptransduc junctions‐ be‐ mechanicaltion mechanical strains strains into biological into biological stimuli. stimuli. LM data LM alsodata demonstrated also demonstrated that the that number the num of‐ tweenber osteocytes of viable osteocytes increases is higherin answer in loaded to bonesthe mechanical than in the control load unloadedapplied ones;to bone TEM segments placedanalyses in cultures. showed This that isthe in intermittent line with compressivethe suggestion loading that not gap only junctions exerts a trophic play func an important‐ role bothtion onin osteocytes,cell‐to‐cell probably communications improving the and fluid in flows cell inside synchronization, the canalicular networkenabling [64], small mol‐ eculesbut to alsobe exchangedstimulates their between metabolic the activity, coupled particularly cells [11,14–16,63]; their protein and/or it is alsoglycoprotein in line with the fact thatsynthesis, osteocytes as shown are byconsidered the increment as mechanoreceptors,of rough endoplasmic ablereticulum, to give thus rise suggesting to the transduc ‐ that these molecules are secreted in answer to mechanical stimuli. tion mechanical strains into biological stimuli. LM data also demonstrated that the num‐ ber of viable osteocytes is higher in loaded bones than in the control unloaded ones; TEM analyses showed that the intermittent compressive loading not only exerts a trophic func‐ tion on osteocytes, probably improving the fluid flows inside the canalicular network [64], but also stimulates their metabolic activity, particularly their protein and/or glycoprotein synthesis, as shown by the increment of rough endoplasmic reticulum, thus suggesting that these molecules are secreted in answer to mechanical stimuli. J. Funct. Morphol. Kinesiol. 2021, 6, 28 10 of 17

viable osteocytes is higher in loaded bones than in the control unloaded ones; TEM analyses showed that the intermittent compressive loading not only exerts a trophic function on osteocytes, probably improving the fluid flows inside the canalicular network [64], but also J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 10 of 18 stimulates their metabolic activity, particularly their protein and/or glycoprotein synthesis, as shown by the increment of rough endoplasmic reticulum, thus suggesting that these molecules are secreted in answer to mechanical stimuli. Moreover,Moreover, Rubinacci Rubinacci and and coworkers coworkers [42 [42]] showed showed that that the the damage-generated damage‐generated ionic ionic cur- rentscurrents they they observed observed in the in cortexthe cortex of frog of frog metatarsal metatarsal bones bones had ahad cellular a cellular origin. origin. Metatarsal Meta‐ bonestarsal bones of adult of amphibianadult amphibian were purposelywere purposely selected selected for the for study the study because because their their shafts shafts (unlike(unlike the the mammalian mammalian one) one) contain contain only only the the osteocyte-bone osteocyte‐bone lining lining cell cell system, system, thus thus giving giv‐ theing firstthe first convincing convincing evidence evidence that that damage-generated damage‐generated ionic ionic currents currents are generatedare generated by the by osteocyte-bonethe osteocyte‐bone lining lining cell system, cell system, i.e., the i.e., actual the actual responsible responsible “entity” “entity” of bone of adaptationbone adapta to‐ bothtion to mechanical both mechanical loading andloading mineral and homeostasis. mineral homeostasis. Later, the Later, same authors the same [43 authors] confirmed [43] theconfirmed observations the observations also in murine also models. in murine models. RecentlyRecently [[58],58], asas farfar asas thethe bonebone adaptation-relatedadaptation‐related cell-signalingcell‐signaling isis concerned,concerned, bothboth Erk1/2Erk1/2 and and Akt Akt were showed to be hyperphosphorylatedhyperphosphorylated in frog longlong bonesbones ofof stressedstressed samplessamples (forced(forced swimming) swimming) suggesting suggesting that that among among the the putative putative osteocyte osteocyte signal signal transduc- trans‐ tionduction mechanisms, mechanisms, Akt signalingAkt signaling is boosted is boosted by increased by increased mechanical mechanical stresses. stresses. Moreover, Moreo the‐ authorsver, the confirmedauthors confirmed again that again the increase that the of increase osteocyte of gap osteocyte junction gap number junction is dependent number is ondependent mechanical on mechanical loading (Figure loading 10). (Figure 10).

FigureFigure 10. TEMTEM micrograph micrograph panel panel representative representative of of the the abundance abundance of ofgap gap junctions junctions (A– (EA) –withE) with respectrespect to simple simple contacts contacts (F (F) )in in overloaded overloaded frogs. frogs. The The head head-arrow‐arrow in ( inA) (indicatesA) indicates the tip the of tip a cy of‐ a toplasmic process protruding in a cell body of an adjacent osteocyte showing an “invaginated fin‐ cytoplasmic process protruding in a cell body of an adjacent osteocyte showing an “invaginated ger‐like” junction. Samples in (B) and (F) are decalcified. ﬁnger-like” junction. Samples in (B) and (F) are decalciﬁed.

5.5. Bone Remodeling and Sclerostin InvolvementInvolvement TheThe bonebone remodelingremodeling processprocess is characterizedcharacterized by distinct phases [[65,66]65,66] (Figure 1111).). DuringDuring unloading,unloading, or or when when sensitivity sensitivity to to strain strain is alteredis altered by by hormones hormones (such (such as parathyroid as parathy‐ hormone,roid hormone, estrogens, estrogens, etc.), etc.), osteocytes osteocytes stop stop producing producing the ionic the ionic currents currents maintaining maintaining the restingthe resting phase, phase, thus thus allowing allowing the the bone bone lining lining cells, cells, the the stromal stromal cells cells and, and, above above all, all, thethe osteocytesosteocytes (the(the mostmost sensitivesensitive onesones toto loadingloading changes)changes) toto produceproduce RANKL,RANKL, thatthat activatesactivates thethe osteoclastogenesis (ﬁrst(first phasephase resorption).resorption). During bone resorption, BBCS is destroyed, butbut it is is likely likely that that surviving surviving overstrained overstrained osteocytes osteocytes are are involved involved in crucial in crucial signaling signaling that thatstops stops osteoclast osteoclast activity activity and triggers and triggers the reversal the reversal phase (second phase (second phase reversion). phase reversion). Various authors believe that the cells of the reversal phase could be involved in sending or receiv‐ ing these signals [67–69]. Other signaling pathways may include matrix‐derived factors such as bone morphogenic protein (BMP)‐2, transforming growth factor (TGF)‐β, and in‐ sulin‐like growth factor (IGF) [66,70,71]. The cells of the reversal phase (probably of stro‐ mal‐fibroblast origin) differentiate into osteoblasts, thus bone formation occurs (third phase deposition). During bone deposition, BBCS is progressively renewed and osteoblast J. Funct. Morphol. Kinesiol. 2021, 6, 28 11 of 17

Various authors believe that the cells of the reversal phase could be involved in sending or receiving these signals [67–69]. Other signaling pathways may include matrix-derived factors such as bone morphogenic protein (BMP)-2, transforming growth factor (TGF)-β, J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 11 of 18 and insulin-like growth factor (IGF) [66,70,71]. The cells of the reversal phase (probably of stromal-ﬁbroblast origin) differentiate into osteoblasts, thus bone formation occurs (third phase deposition). During bone deposition, BBCS is progressively renewed and osteoblast activityactivity stops stops when when local local physiological physiological loading loading conditions conditions are are restored; restored; the the osteocytes osteocytes in in thethe newly-laid-down newly‐laid‐down bone bone matrix matrix produce produce again again the the steady steady ionic ionic currents currents that that return return the the bonebone to to the the resting resting phase, phase, therefore therefore halting halting osteoblast osteoblast activity. activity.

Figure 11. Schematic drawing of postulated BBCS role during bone remodeling cycle. See text for Figure 11. Schematic drawing of postulated BBCS role during bone remodeling cycle. See text explanation. for explanation.

AsAs far far as as the the regulation regulation of of bone bone remodeling remodeling process process is is concerned, concerned, it it has has recently recently been been shownshown that that sclerostin sclerostin has has a relevanta relevant role, role, displaying displaying both both autocrine autocrine and and paracrine paracrine effects effects [66]. Sclerostin[66]. Sclerostin (a 22-kDa (a 22 glycoprotein),‐kDa glycoprotein), due to due SOST to promoterSOST promoter hypomethylation hypomethylation [72,73], [72,73],is cur- rentlyis currently considered considered the major the mediatormajor mediator of the molecularof the molecular osteocyte osteocyte mechanisms mechanisms involved in‐ involved the process in the ofprocess adaptive of adaptive bone responses. bone responses. In the mature In the skeleton,mature skeleton, sclerostin sclerostin is mainly is synthesizedmainly synthesized by differentiated by differentiated mature osteocytes, mature osteocytes, while preosteocytes, while preosteocytes, bone lining bone cells, lining and osteoblastscells, and osteoblasts express very express low levels very oflow sclerostin. levels of The sclerostin. central The role central of sclerostin role of is sclerostin performed is throughperformed the through interplay the between interplay two between opposing two mechanisms: opposing mechanisms: (1) unloading-induced-high (1) unloading‐in‐ sclerostinduced‐high levels, sclerostin that antagonize levels, that canonical antagonize Wnt canonical in osteocytes Wnt andin osteocytes osteoblasts and and osteoblasts promote non-canonicaland promote non Wnt‐canonical and/or other Wnt and/or pathways other in pathways osteocytes in and osteocytes osteoclasts and [ osteoclasts74–76]; (2) me-[74– chanical76]; (2) mechanical loading-induced-low loading‐induced sclerostin‐low levels, sclerostin that levels, activate that Wnt-canonical activate Wnt signaling‐canonical and sig‐ bonenaling formation. and bone Thus,formation. adaptive Thus, bone adaptive remodeling, bone remodeling, occurring inoccurring different in bone different compart- bone compartments, is driven by altered sclerostin levels, which regulate the expression of the other osteocyte‐specific proteins, such as RANKL, its decoy receptor osteoprotegerin (OPG), and other proteins (Figure 12). Under the regulation of differential RANKL and OPG production, due to specific conditions, sclerostin creates a dynamic RANKL/OPG ratio [77–79], leading to either bone resorption or formation. Such opposite up‐ or down‐ regulation of the remodeling phases allows osteocytes (i.e., the permanent bone cells) to function as “the orchestrators” of osteoclasts and osteoblasts (i.e., the transient operating J. Funct. Morphol. Kinesiol. 2021, 6, 28 12 of 17

ments, is driven by altered sclerostin levels, which regulate the expression of the other osteocyte-speciﬁc proteins, such as RANKL, its decoy receptor osteoprotegerin (OPG), and other proteins (Figure 12). Under the regulation of differential RANKL and OPG production, due to speciﬁc conditions, sclerostin creates a dynamic RANKL/OPG ratio [77–79], J. Funct. Morphol. Kinesiol. 2021, 6, x FOR PEER REVIEW 12 of 18 leading to either bone resorption or formation. Such opposite up- or down-regulation of the remodeling phases allows osteocytes (i.e., the permanent bone cells) to function as “the orchestrators” of osteoclasts and osteoblasts (i.e., the transient operating bone cells) ensuringbone cells) the ensuring transition the from transition bone resorption from bone to resorption bone formation. to bone In formation. this context, In the this putative context, inhibitionthe putative of sclerostin inhibition represents of sclerostin a strategy represents to target a strategy bone remodelingto target bone unbalance remodeling in skeletal unbal‐ disorders,ance in skeletal as osteoporosis, disorders, as rheumatoid osteoporosis, arthritis, rheumatoid bone related-genetic arthritis, bone disorders, related‐genetic notwith- dis‐ standingorders, notwithstanding further studies arefurther needed studies to better are needed clarify how to better sclerostin-Ab clarify how modulates sclerostin bone‐Ab resorptionmodulates since bone some resorption authors reportedsince some the lackauthors of modulation reported the of RANKL, lack of OPG,modulation and other of regulatorsRANKL, OPG, of osteoclastogenesis and other regulators during of sclerostin-Abosteoclastogenesis treatment during [80 sclerostin–82]. Recently,‐Ab treatment various papers[80–82]. were Recently, published various on the papers topic were where published new therapeutic on the topic strategies where were new proposed, therapeutic as denosumab,strategies were rosomozumab, proposed, as etc. denosumab, [83–86]. rosomozumab, etc. [83–86].

Figure 12. Scheme of RANK/RANKL/OPG system. Under specific conditions, differential RANKL Figure 12. Scheme of RANK/RANKL/OPG system. Under speciﬁc conditions, differential RANKL and OPG production are induced, and the dynamic RANKL/OPG ratio leads to either bone for‐ and OPG production are induced, and the dynamic RANKL/OPG ratio leads to either bone formation mation or bone resorption. or bone resorption.

6.6. InterplayInterplay betweenbetween Mineral Mineral and and Skeletal Skeletal Homeostasis Homeostasis and and Osteocyte Osteocyte Role Role Mediated Mediated byby Sclerostin Sclerostin BoneBone remodeling remodeling is is the the main main tool tool by by which which the the skeleton skeleton answers answers to both to both the metabolic the meta‐ andbolic mechanical and mechanical demands, demands, thus thus regulating regulating the the mineral mineral and and skeletal skeletal homeostasis. homeostasis. The The mineralmineral homeostasishomeostasis keepskeeps inin relativelyrelatively stablestable balancebalance thethe concentrationconcentration ofof mineralmineral ions,ions, asas calciumcalcium and phosphate, in in the the organic organic liquids; liquids; the the skeletal skeletal homeostasis homeostasis allows allows the the ad‐ adjustmentjustment of of shape, shape, mass mass and and bone bone structure structure following following the actual mechanical needsneeds of of the the skeletalskeletal segments.segments. TheThe twotwo processesprocesses areare notnot independent independent ofof each each other; other; in in fact, fact, various various investigationsinvestigations showed showed that that they they are are functionally functionally correlated correlated in in driving driving thethe bonebone responses,responses, asas a a whole, whole, to to different different experimental experimental conditions conditions [ 87[87–91].–91]. Here,Here, the the authors authors want want to to stress stress the the importance importance of of the the interplay interplay between between mineral mineral and and skeletalskeletal homeostasis homeostasis (i.e., (i.e., a a dynamic dynamic balance balance of of the the two two homeostases) homeostases) in in modulating modulating and and guiding bone’s response to dietary/metabolic alterations and/or unbalance of loading con‐ ditions. In particular, it is important to be emphasized that mineral homeostasis involves bone response to a variation of mineral serum levels (in consequence of various condi‐ tions, such as hormonal alterations or dietary regimen), while skeletal homeostasis im‐ plies bone answers to loading modifications. In metabolic osteoporosis due to a calcium‐deprived diet for one month in a rat model, Ferretti and coworkers [86] showed that the lack of calcium in the diet does not J. Funct. Morphol. Kinesiol. 2021, 6, 28 13 of 17

guiding bone’s response to dietary/metabolic alterations and/or unbalance of loading conditions. In particular, it is important to be emphasized that mineral homeostasis involves bone response to a variation of mineral serum levels (in consequence of various conditions, such as hormonal alterations or dietary regimen), while skeletal homeostasis implies bone answers to loading modifications. In metabolic osteoporosis due to a calcium-deprived diet for one month in a rat model, Ferretti and coworkers [86] showed that the lack of calcium in the diet does not lead to a unique bone answer, since the interplay between mineral and skeletal homeostasis influence the bone loss in different sites of the two bony architectures (trabecular versus cortical bone) in both axial (lumbar vertebrae) and appendicular (femurs) skeleton. Despite the observed reduction of trabecular number (due to the maintenance of mineral homeostasis), an intense activity of bone deposition occurs on the surface of the few remaining overloaded trabeculae (in answering to mechanical stresses and, consequently, to maintain the skeletal homeostasis). The authors also reported: (i) the evidence that the more involved bony architecture is the trabecular one, that is the most susceptible to dynamic homeostasis variations; (ii) the different responses recorded in different sites of cortical bone are dependent on their main function in answering mineral and/or skeletal homeostasis. Moreover, on the basis of evidence that PTH(1-34) improves recovery of bone fragility and accelerates bone healing [92–98], a recent study, performed by Ferretti and coworkers in animal models [91], showed that, after one month of calcium-free feeding, the normal diet restoration with/without concomitant PTH (1-34) administration differently determines the pattern of bone mass recovery, in terms of amounts and sites, not only depending on the mineral homeostasis but also under the influence of the skeletal homeostasis. This study highlighted (i) the importance of the interplay between mineral and skeletal homeostasis, (ii) the evidence that the more involved bony architecture, in modulating and guiding bone’s response to dietary/metabolic alterations, is again the trabecular bone, (iii) the trabecular bone as the preferential target of PTH (1-34). These observations agree with those of various authors [99–101] showing that appendicular skeleton (mostly concerning cortical bone) answers mainly to mechanical demands (i.e., is devoted to the skeletal homeostasis) while axial skeleton (mostly concerning trabecular bone) answers mainly to metabolic demand (i.e., is mainly devoted to the mineral homeostasis). With regard to the osteocyte’s role in mediating the interaction between the two types of homeostasis, it represents (as previously reported) both the bone mechano-sensor and the major producer of some signaling proteins. As previously mentioned, one of the most investigated osteocyte proteins is sclerostin, that inhibits osteoblast activity, depending (among other factors/conditions) on the answers to mineral and skeletal homeostasis. In this context, it is not surprising that sclerostin expression is higher in animals fed a calcium-free diet with respect to all animals with restored normal diet and to the control groups [90], also depending on the answers to mineral and skeletal homeostasis [102].

7. Conclusions In conclusion, despite the “segregation” within the mineralized bone matrix, the osteocyte is the dynamic bone cellular element that triggers/guides/modulates a series of sophisticated and interconnected processes, in hierarchic priority: their balance is allowed by osteocyte capability to sense the different bone demands (i.e., metabolic, hormonal, mechanical, etc.) and, depending on the interaction with the actual systemic conditions, to act on the “operator” bone cells that form and destroyed bone tissue under the direction of the best orchestrator.

Author Contributions: Both authors have written the text and agreed to the published version of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. J. Funct. Morphol. Kinesiol. 2021, 6, 28 14 of 17

Informed Consent Statement: Not applicable. Data Availability Statement: Data contained within the review are available in the papers cited in the section References. Acknowledgments: The authors thank Marta Benincasa for her valuable help in setting iconography. Some recent data from author’s Lab, reported in the Review, were supported by funds “Department of Excellence 2018–2021” (Department of Biomedical, Metabolic and Neural Sciences). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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