Bone tissue physiologically undergoes a remodeling which is the result of a dynamic balance between bone formation by osteoblasts (OB) and resorption by osteoclasts (OC). These antagonistic processes are essential to support body weight and maintain its multiple physiological activities. Moreover, bone formation and resorption allow for response to dynamic mechanical forces and also assist in functional and metabolic demands. Nevertheless, in some specific conditions, homeostasis at rest can be altered by physiological stimuli as observed during exercise and load, but also associated with pathologies such as aging-related osteoporosis, metabolic diseases, cancer, and therapeutic interventions ^[1][2][1,2].

In particular, three stressors seem to prevail in bone remodeling: mechanical stress, oxidative imbalance, and acidosis. The identification of molecular sensors, transducers, and signaling pathways involved in these processes is of paramount importance in order to design more efficient clinical, pharmacological, and nutritional strategies, as well as to improve wellness through a healthy lifestyle and motor activity. Indeed, the promotion of physical exercise during growth increases the chances of strengthening bone and delaying the osteoporotic insurgence associated with higher bone mineral density and lower fracture incidence, with beneficial effects maintained in adulthood ^{[3][4][5][6][7]}[3,4,5,6,7]. Moreover, exercise duration, type, and intensity drive exercise-induced inflammatory and metabolic responses that concur to improve bone remodeling [8].

2. The Role of Mechanical Stress, Oxidative Imbalance, and Acidosis in Bone Remodeling

2.1. Mechanical Stress

Bone load is necessary to maintain and control bone mass, growth, and remodeling, as originally noted by Galileo, later confirmed by Wolff’s law (1892), and further stated by Harold Frost in the 1980s ‘mechanostat’ concept. The formation, resorption, and adaptation of the skeletal system depend on the forces applied: in fact, the bone is weakened in their absence, as occurs in disuse osteoporosis which increases the incidence of fracture ^[9][83]. The cellular components of bones include osteoblasts (OB), osteoclasts (OC), osteocytes, as well as their progenitor stem cells; all of them display different sensitivity to shear stress, hydrostatic pressure, mechanical stretch, and tension, together with matrix stiffness and alignment ^[10][84]. The most abundant population is represented by osteocytes, which respond to mechanical stimuli and propagate information to other cells in autocrine and paracrine modes ^[9][83]. During mechanical loading, osteocytes sense fluid flow (FF) in the lacunar-canalicular system while bone marrow mesenchymal stem cells (BMSC) in the medullary cavity ^[10][84]: these events drive BMSC proliferation and differentiation, altering their morphology, volume, and cytoskeleton arrangement and enhancing osteogenic gene expression. In the same cell type, oscillatory FF (pressure 1–5 Pa, frequency 0.5–2 Hz, duration 1–4 h) triggers the recruitment of differential gene patterns according to the intensity, frequency, and duration of shear stress. In particular, in the early stages of osteogenesis, short-term stimulation upregulates osteogenic markers such as Cox2 (cycloxygenase), Opn (osteopontin, a component of the extracellular matrix ECM), and Runx2 (transcription factor), while long-term treatment enhances collagen and matrix formation in late phases ^[10][11][84,85]. In addition to the externally applied mechanical load, the intrinsic mechanical properties of ECM strongly affect all cell bone types. For instance, ECM stiffness redirects MSC from adipogenic to osteogenic fate: the control of stem cell osteogenesis could be mediated by microtubule dynamics and deformation involving the microtubule-associated protein DCAMKL1 ^[10][12][84,86]. In summary, mechanical stresses exert multiple effects on bone cells: on one hand they promote morphological and cytoskeletal changes, on the other they alter tissue-specific gene expression.

2.2. Oxidative Imbalance

Oxidative stress (OS) is the imbalance of the oxidative state established by cellular pro- and anti-oxidant agents; it plays an important and widespread role in the physiopathology of many tissues as well as in senescence ^[13][87]. Aerobic cellular metabolism physiologically leads to the potentially threatening accumulation of reactive oxygen species (ROS) and free radicals (superoxide anion, hydrogen peroxide, hydroxyl radical) which are counteracted by antioxidants which act as scavengers and cell detoxifiers, dampening the overall damage. This is a common mechanism observed during aging and closely related to the development of osteoporosis. Indeed, upon persistent OS, a positive self-regenerating loop is produced leading to an array of pathophysiological changes ^[14][88]. Nonetheless, ROS are required for a wide range of healthy functions. In particular, they act as signaling molecules capable of modulating cell proliferation and differentiation, immune cell responses, and stem cell renewal ^[15][89]. In bone tissue, ROS physiologically enhance OC differentiation with the involvement of the RANKL pathway ^[16][90]. The redox state should be maintained in a precise balance to preserve the correct bone remodeling, since an increase in OC activity can easily lead to ROS accumulation and cellular stress. Indeed, ROS foster apoptosis of OB and osteocytes, thus promoting osteoclastogenesis and inhibiting mineralization and osteogenesis. The resulting redox dysregulation is involved in the pathogenesis of bone loss and can be observed, among the others, in osteoporosis.

2.3. Acidosis

The homeostatic imbalance between osteoclast-mediated resorption and OB-dependent bone formation is responsible for the insurgence of osteoporosis, an age-related skeletal disease characterized by decreased bone mass and altered architecture, leading to an increased risk of fragile fractures commonly found in the elderly. In addition to a high overall OS state, extracellular acidity is a paramount contributor to osteoporotic progression. The protons secreted by OC during bone resorption physiologically lead to the OC-bone interface acidification which interferes with bone cell biology. While an alkaline pH promotes mineralization and osteoblastic potential, acidosis in turn stimulates osteoclastic resorption. The OC sensitivity to protons involves early expression of the ovarian cancer G-protein-coupled receptor 1 (OGR1) which is responsive to H⁺ fluxes and is thought to play a role in osteoclastogenesis ^[17][18][103,104]. Acidic environment can also induce autophagy in OB ^[19][105] and inhibits OB-mediated biomineralization by interfering with ALP activity and impairing collagen proteins osteopontin and osteocalcin ^[20][106]. With aging, blood H⁺ levels increase and bicarbonate concentration is depressed, indicating progressive worsening low-level metabolic acidosis. Finally, it is worth noting that, as already noted, excessive skeletal loading is usually related to inflammatory processes and acidosis.

3. Ion Channels as Membrane Multi-Sensors and Transducers in Bone Remodeling

3.1. Piezo

The mammalian Piezo family includes Piezo1 and 2 proteins which arrange in trimers acting as MS calcium-permeable channels. The physiological activation of Piezo follows the physical deformation of the lipid bilayer resulting from a finely tuned modulation by intracellular tethers of the cytoskeleton and by extracellular matrix ^{[9][21][22][23][24]}[83,115,117,118,119]. This event can be mimicked in artificial lipid bilayers as well as by pharmacological treatments ^[25][116]. Although the mechano-gating is the most attractive feature of Piezo, they can also act as sensors for other biologically relevant variables, as revealed by patch clamp evidence reporting their inhibition by extracellular acidification below pH 6.9 ^[26][21]. Piezo1 is required for mammalian embryonic development and is mainly found in hollow organs, such as lungs, blood vessels, bladder, and gastrointestinal tract, but its recruitment is also reported in OB, osteocytes and chondrocytes thereby regulating osteogenesis and cartilage degradation in joints ^[27][28][18,122]. Similarly, Piezo1 regulates MSC fate thereby triggering osteoblastic differentiation and hindering the adipogenic one ^[29][17]. As aforementioned, the most relevant stressor modulating Piezo1 is the mechanical force (hence the name), which is a key factor in bone remodeling. In order to investigate its downstream intracellular targets, Piezo1 was activated with its specific agonist Yoda1 in MSC leading to the identification of ERK1/2 and p38 signaling as key pathway in correlation with Bone Morphogenetic Protein 2 (BMP2), a critical osteogenic growth factor ^[29][17]. The same effect can be obtained upon hydrostatic pressure application ^[29][30][17,123]. Piezo1 is also detected in osteocytes, which are the most abundant bone cells, where mechano-transduction is mediated by Piezo1-Akt signaling and suppression of sclerostin, an important regulator of bone formation ^[31][124]. Accordingly, the ablation of this channel in KO mice compromises bone responses to mechanical forces ^[32][20]. In addition to its physiological functions, a contribution of Piezo1 has also been reported in some bone diseases. In particular, its downregulation is related to osteoblast dysfunction in osteoporotic patients, opening the possibility for new selective therapeutic approaches ^[27][18]. A quantitative computed tomography (μ-QCT) study performed in Piezo1-deficient mice of different ages revealed that the newborn mice show no differences with wt animals ^[33][125]. The lack of effect in neonatal age could be explained by the absence of mechanical stimuli activating the channel, again strenghtening the hypothesis for its involvement in bone tissue mechano-sensing and transduction ^[33][125].

3.2. TRP

Transient Receptor Potential channels (TRP) are a class of cationic ion channels grouped into six subfamilies (TRPC, TRMPV, TRPM, TRPA, TRPL and TRPP) according to sequence homology. They are composed of six membrane-spanning helices that usually organize in functional homo- or heterotetramers and can be regulated or modulated by a number of stressors, including heat, pressure, tension, shear stress, oxidative stress and hypoxia. TRP are ubiquitously and redundantly distributed in healthy and altered human tissues where they play a large variety of functions ^[34][128].

3.2.1. TRPV4

TRPV4 is a non-selective polymodal calcium-permeable channel widely expressed throughout the body and recruited in both physiological and pathological events. In bone tissue, TRPV4 has been associated with OC and OC differentiation, pathogenesis of osteoporosis and other metabolic bone diseases ^[35][131]. Its pore gating may be triggered by mechanical stress, temperature, or hypoosmolarity. TRPV4 exhibits mechano-sensitivity. However, whether it fulfills the criteria for true MS channels has been debated ^[21][115]. Several mechanisms may mediate its mechano-activation, involving Phospholipase A2 (PLA2) and Phospholipase C (PLC). Upon osmotic swelling or shear stress exposure, PLA2 binds membrane phospholipids and releases arachidonic acid (AA), the fatty acid precursor of eicosanoids that include cytochrome p450-related epoxyeicosatrienoic acid (EET). Both AA and EET act as TRPV4 modulators ^{[36][37][38][39]}[132,133,134,135]. The mechano-sensitivity of TRPV4 is detected in many cell bone subtypes, possibly interacting with other molecular machineries. In mouse osteoblastic cells, hypo-osmotic stress increases intracellular calcium through TRPV4 and TRPM3 to regulate RANKL and NFATc1 expression ^[40][25]. An important role is played by the primary cilium, an antenna-like, not motile structure that extends from the surface of most mammalian cell types into the extracellular space, where TRPV4 preferentially localizes in MSC osteoblast precursors and in differentiated osteocytes ^[41][42][22,31]. Chondrocytes produce and maintain the articular cartilage by sensing and responding to changing mechanical loads; TRPV4 and Piezo are key actors in these physiological events and mediate mechanical and inflammatory signals ^[43][136].  Mechanical stimulation of TRPV4 has also been associated with osteoporosis due to its impact on OC survival ^[44][27] and the higher osteoporotic fracture risk related to TRPV4 deficiency ^[45][137]. In particular, and similarly to what was previously discussed for OB ^[40][25], OC differentiation enhanced by the channel involves the calcium-calcineurin-transcription of NFATc1 signaling pathway which promotes osteoclastogenesis-related cFos and TRAP gene transcription ^[44][27]. Both Piezo1 and TRPV4 have been detected in OB and their co-expression suggests a MS capability ^[46][26]. Interestingly, however, these two channels are unable to respond separately, suggesting the requirement of a coupling mechanism. Furthermore, brief shear stress induced by fluid flow selectively activates Piezo1 (but not TRPV4) raising intracellular calcium levels and recruiting PLA2. This event in turn triggers the opening of TRPV4, which is responsible for a second phase of Ca²⁺ influx ultimately leading to pathological events ^[47][138].

3.2.2. TRPA1

TRPA1 is the only member of the TRPA group. It is a polymodal channel sensitive to tissue damage, noxious cold, endogenous compounds released by oxidative reactions, as well as to pro-inflammatory peptide bradykinin via the PLC signaling ^[48][142]. It is distributed in a large variety of cell types often in association with TRPV1, especially in nervous tissues. Consistently, the combination of these two channels is considered relevant in nociception, but also in the inflammatory milieu ^[49][143]. The evidence of TRPA1 expression in bone tissue is controversial; no transcriptional levels have been detected in vitro in OB and OC cell lines, while it is identified in MSC OB precursors ^[50][43], in bone from a breast cancer pain mice model ^[51][46] in periodontal ligament cells ^[52][42] and odontoblast-like cells ^[53][44]. Due to its multimodal gating and peculiar sensitivity to oxidative stress and inflammation, this protein should deserve future investigation in bone biology. TRPA1 has previously been proposed as a potential primary MS calcium channel within the mammalian sensory nervous system ^[54][144], although recent models suggest a major role for the transmembrane channel-like proteins (TMC1 and TMC2) and transmembrane proteins (TMEM). TRPA1 recruitment by membrane stress has been observed using chemical agents and hyperosmotic solutions ^[55][47]. In addition, new light on its controversial activity mechano-sensor has been shed by studies in artificial lipid bilayers where the thiol reducing agent TCEP is able to abolish TRPA1 activity, suggesting an intrinsic mechanosensitivity dependent on the redox state ^[55][47]. TRPA1 has also been found in periodontal ligament (hPDL) cells; upon mechanical stimulation, hPDL selectively upregulate the protein (but not other mechanoreceptors), possibly supporting its functional role: the downstream pathway involves the phosphorylation of MAPKs ERK1/2, p38 and JNK ^[52][42]. In addition, TRPA1-mechano-activation upregulates CCL2, a chemokine ligand involved in osteoclastogenesis, in periodontal ligament cells ^[52][56][41,42]. Odontoblast-like cells co-express TRPA1 and TRPV4 that is known for its sensitivity to mechanical stretch ^[53][44]. The involvement of both these channels follows hypotonicity-induced membrane elongation and involves p38 MAPK downstream pathway: interestingly, treatment with the pro-inflammatory peptide TNFα upregulates TRPA1 and downregulates TRPV4 ^[53][44].

3.2.3. TRPM7

Another TRP channel involved in bone metabolism is TRPM7, whose deletion causes embryonic lethality in mice ^[57][149]. TRPM7 is a cation channel (Ca²⁺ and Mg²⁺ permeant) covalently linked to a protein kinase domain: it is ubiquitously distributed throughout the body acting as a regulator of Mg²⁺ homeostasis, motility, and proliferation. Odontoblasts, the dentin forming cells capable of sensing mechanical stimulation, express predominantly TRPM7 ^[58][52] among the numerous MS TRP (TRPA1, TRPC1, TRPC6, TRPV1, TRPV4). Based on a careful pharmacological approach, Won et al. demonstrated that TRPM7 is a fundamental mechano-sensor facilitating intracellular Ca²⁺ signaling in odontoblastic process ^[59][53]. The importance of TRPM7 in dental mineralized tissues has been further proved by its upregulation during amelogenesis in ameloblasts and odontoblasts, while TRPM7 kinase-inactive knock-in mutant mice are affected by reduced enamel mineralization and weakened enamel structure, albeit independently of ion channel function ^[60][150]. To assess the role of TRPM7 in bone development, the same group ^[61][50] generated rx1-Cre-dependent Trpm7 mesenchymal-deleted mice and observed shortened bones and impaired trabecular bone formation, pointing out a possible impairment of chondrogenesis. The scautholars concluded that TRPM7 is critical as a cation channel rather than as a kinase in bone development.

3.3. ASIC/ENaC

The acid-sensing ion channels (ASIC) and epithelial sodium channels (ENaC) are members of a family of proteins that play critical roles in mechanosensation, chemosensation, nociception, and regulation of blood volume and pressure ^[62][151]. They form hetero- or homotrimers with subunits that share a common structure with two transmembrane and a large extracellular loop.

3.3.1. ASIC

Acid-sensing ion channels (ASIC) are a group of proton-gated cation-permeable channels that belong to the family of the degenerin Deg/EnaC group ^{[21][63][64][65]}[55,115,152,153] and are found in adult BMSC-derived OB ^[66][56]. Culturing OB in media with increasing pH, reduced osteoblastogenesis was observed under acidic conditions concomitant with a progressive upregulation of ASIC2, ASIC3, and ASIC4 ^[63][55]. This pH-dependent pattern could support a role of ASIC in the pathogenesis of osteoporosis, which is characterized by a sharply acidic pH responsible for osteoblastogenesis impairment. ASIC2 is enhanced in human bone cells from osteoporotic vertebral fractures and depressed during osteoblast differentiation and mineralization ^[63][55].

3.3.2. ENaC

The amiloride-sensitive epithelial sodium channel (ENaC) is a major contributor to intracellular sodium homeostasis; the αENaC subunit is found in skeletal cells including articular chondrocytes and OB where it has been proposed to contribute to mechanotransduction, sodium transport and extracellular sodium sensing. Interestingly, a correlation between bone Na⁺ content and bone disease has been reported, suggesting that ENaC-mediated Na⁺ regulation may influence osteogenesis ^[67][68][69][60,61,159]. UMR-106 OB-like cell line and human OB in primary culture express αENaC subunit ^[70][59]. Bovine α-ENaC exhibits a pressure-induced activation in lipid bilayers and when subjected to a hydrostatic pressure gradient ^[71][160]. Accordingly, reconstitution of rat a-ENaC in LM(TK2) cells, a null cell for stretch-activated, nonselective cation channels, provides a mechanically-gated channel permeable to sodium and calcium ions; elegant patch clamp experiments describe its biophysical properties upon application of negative pressure, cell swelling, or patch excision ^[70][59].

3.4. P2X Purinergic Receptors

P2X purinergic receptors (P2XR) are ligand-gated cationic channels primarily activated by extracellular ATP (eATP). The two-spanning transmembrane domain subunits associate into functional homo- or heterotrimers complexes structurally similar to ASIC/EnaC. P2XR are sensitive to different extracellular stimuli including biologically relevant inorganic ions, such as extracellular protons (P2XR share some structural similarities with ASIC), Zn²⁺ and Ca²⁺, that allosterically modulate channel activity ^{[72][73][74][75]}[161,162,163,164]. Furthermore, hypoxia interferes with P2XR expression and membrane targeting both in physiological and pathological conditions ^[76][111]. Some P2XR are found in bone cells, and OC in particular show the highest variety of isoforms. P2X7R seems to be the prevalent member, contributing to normal and pathological bone metabolism with a key contribution to the early stages of bone repair; its deficiency leads to bone loss and susceptibility to fractures ^[77][70]. In addition, it is also involved in OB differentiation and osteogenesis ^[78][79][80][66,67,165]. P2X7R knockout results in impaired bone formation and lower mechano-sensitivity in vivo ^[80][165]. The molecular mechanism underlying mechano-sensitivity of P2X7 was explored in MSC subjected to physiological and supra-physiological mechanical loading; early expression of mechano-related genes such as c-Jun, c-Fos, and nuclear factor-κB was observed together with increased levels of eATP and the release of pro-osteoclastogenic factors; consequently, the overall OC number was increased ^[81][64]. P2X7R is the most abundant P2X form in OC and affects their differentiation ^[81][64]. It has been associated with osteoporotic progression through an active involvement in OB-to-OC signaling, where it supports OC survival. Consistently, its absence results in increased bone loss, due to a significant OB loss and OC increase, thus leading to an imbalance that explains the observed excessive bone resorption ^[82][68]. The potential causes of P2X7R loss have been investigated in some studies which have highlighted the occurrence of single nucleotide polymorphisms (SNP) possibly correlated with osteoporosis incidence; they can be distinguished between loss-of-function SNP that are associated with bone mineral density (BMD) decrease and gain-of-function SNP that reduce the risk of disease development ^[83][84][168,169]. 

3.5. Connexins and Pannexins

Connexins (Cx) and Pannexins (Panx) share a similar structure with four transmembrane domain subunits arranged in functional examers ^[85][170]. The Panx family is relatively small and consists of three members (Panx-1-3) while Cx group includes more than 20 proteins ^[86][79]. Connexins assemble at the level of the plasma membrane in oligomeric complexes that play critical roles in many cellular processes including messenger transport, cell coupling, morphogenesis, differentiation, and growth in a wide variety of tissues. They are physiologically activated by mechanical stimulation, showing also sensitivity to pH and to the reduction of extracellular calcium concentration. In addition, Cx43 and Cx50 are also recruited by OS and other forms are regulated by phosphorylation ^[87][88][89][171,172,173]. Unlike Cx, Pannexins do not form cell–cell channels but act mainly as single-membrane channels ^[90][174]. However, they share some functions with the Cx family such as the involvement in Ca²⁺ signaling ^[91][175]. Of the three members of PanX family, Panx1 is by far the most studied. Its activity is mediated by several mechanisms including stretch, K⁺ and Ca²⁺ currents, phosphorylation and C-tail cleavage ^[92][77]. Mechanical activation of Panx1 triggers intracellular calcium waves and the extracellular release of ATP ^[93][176]; therefore, it is often associated with purinergic signaling involved in many processes such as apoptosis, blood pressure regulation, neuropathic pain, and excitotoxicity. Panx1 is also sensitive to changes in extracellular pH; alkaline environment induces a slight reduction in Panx1-mediated conductance, while a stronger effect is detected in terms of ethidium uptake (increase with alkalization and decrease with acidification) ^[92][77]. Some Cx and Panx are present in bones and modulate tissue remodeling, as well as osteoprogenitor and chondrocyte cell differentiation ^[86][79].

3.5.1. Connexins

Bone cells are functionally connected in a sort of functional syncytium through the gap junction channels including Cx proteins ^[94][177]. Within this family, Cx43 is one of the most important members for its key roles in bone embryogenesis, cell differentiation and mineralization. Several lines of investigation lead to the conclusion that the non-junctional MS Cx43 hemichannels are essential during the early stages of OB differentiation and maturation, providing an efflux pathway for ATP or prostaglandin E2 (PGE2) ^{[95][96][97][98]}[178,179,180,181]. Cx43 hemichannels are probably not directly mechanosensitive being their response possibly mediated by integrins, as suggested by the ability of fluid flow shear stress to promote the interaction between integrin α-5 and Cx43 and the following PGE2 release from osteocytes ^[99][71].

3.5.2. Pannexins

Panx1 and 3 are involved in the integrity and function of the interconnected network of OB, osteocytes and OC ^{[86][100][101][102][103]}[78,79,80,184,185]. The mechano-sensitivity of Panx1, together with its ability to release ATP, explains its relationship and interaction with P2X7R ^[104][186]. The formation of a functional complex among the two proteins has been described in osteocytes, where it could mediate apoptosis ^[105][106][75,76]. Panx1, P2X7R, and the T-type voltage-operated calcium channel CaV3.2-1 co-localize with integrin β3 on osteocyte processes, likely providing a specialized MS machinery that functions in a distinct manner ^[9][83], ^[107][187]. The complex is recruited following shear stress and osmotic pressure, while P2X7R mechano-transduction seems indirect and is likely mediated by Panx-1-dependent ATP release into the extracellular medium. The accumulated eATP then binds to the low-affinity P2X7R triggering the opening of its pore with an additional influx of calcium and potassium efflux ^[39][108][135,188]. Similar cooperation between ATP and P2X7R has been elucidated in other contexts such as inflammasome assembly and caspase-1 recruitment in monocytes and macrophages ^[109][110][189,190].