The importance of bones for a living organism is undeniable and goes far from just providing structural support for the body, protecting vital organs and exchanging minerals. Bones also comprise a multi-functional system that interacts with other systems and abnormalities in bone tissues may result in mild or severe diseases.

Bone tissue is composed of the bone matrix and five different cell types. The bone matrix contains an inorganic (carbonated hydroxyapatite) and an organic phase (mainly type I collagen and several growth factors) whilst the cellular content of the bone tissue comprises of osteoprogenitors, osteoblasts, osteocytes, osteoclasts and lining cells[1]. Osteoprogenitor cells comprise pluripotent cells of mesenchymal origin, localised on bone surfaces[1] which have the ability, under the appropriate conditions, to commit and differentiate towards osteoblasts[1]. On the same bone osteoblasts, the bone forming cells, are cited. They are responsible for the protein synthesis of the bone matrix as well as its calcification[1]. The cavities of the calcified bone matrix bear osteocytes which comprise entrapped inactive osteoblasts forming a net of communicating cells inside the calcified matrix[1]. Osteoclasts are large multinucleate cells of blood monocyte origin, settled inside bone resorption lacunae and they are responsible for bone resorption in bone remodeling areas[1]. Lining cells comprise inactive osteoblasts with the ability to protect bone surfaces from bone resorption[1].

Runt-related transcription factor 2 (Runx2) or core-binding factor subunit alpha-1 (Cbfα1), the major osteo-specific transcription factor[2] is responsible for the regulation of osteoblast differentiation as well as for hypertrophic cartilage synthesis[2,3]. Its expression is necessary and sufficient for the commitment of mesenchymal cells towards the osteoblastic cell line[4].

Abnormalities in Runx2 expression are indicative of its importance in bone biology. When Runx2 is expressed ectopically it has been shown to lead to increased expression of osteocalcin, alkaline phosphatase, collagenase-3, bone sialoprotein and collagen type Iα1[5]. Osteoblast maturation in mice bearing a mutant runx2 gene is inhibited and thus so are the procedures of intramembranous and endochondral ossification[6,7]. Furthermore, it has been shown that differentiation of stem cells in adipocytes and chondrocytes in runx2 knockout mice has not been impaired. In addition, heterozygous mice (runx2^-/+) developed characteristic skeletal abnormalities similar to human heritable skeletal disorder cleidocranial dysplasia (CCD) abnormalities[8]. On the other hand, tissue-specific Runx2 over-expression in transgenic mice results in decreased bone density, bone fractures and osteopenia[7,9,10].

Bone remodeling, the continuous bone reconstruction is of major importance for conserving bone structural integrity as well as for the bone to perform its metabolic role by modulating calcium and phosphorus levels in the body[1].

Shortly, bone remodeling activation depends mostly on local factors and their effects on mesenchymal progenitor cells. Bone reconstruction initiates with osteoclasts performing bone resorption and forming cavities inside the bone. At the end of this phase, osteoclasts produce the appropriate signals for the initiation of bone synthesis[1]. Osteoblasts quickly cover the cavity surfaces and synthesize new bone. Those two bone remodeling phases, bone formation and resorption are closely correlated and interconnected. This means that under normal conditions, the newly formed and the reabsorbed bone quantities are equal[11]. Impaired bone remodeling may lead in pathophysiological bone conditions like osteoporosis, Paget’s disease, orthopedic disorders and osteopetrosis among others[1].

Research has shown that the GH-IGF-1 axis may also be of significance in the modulation of bone mass quantity and quality. More specifically, growth hormone (GH) is suggested to potentially play a role on bone remodeling[12]. However, the exact mechanisms through which GH acts on osteoblast biology have not been elucidated[12].

The receptor activator for nuclear factor κb (RANK)/ receptor activator for nuclear factor κb ligand (RANKL)/ osteoprotegerin (OPG) system comprises the main modulator of bone remodeling[13]. More specifically, pre-osteoclasts express RANK in their surface. Its ligand, RANKL, is produced in osteoblasts, stromal cells as well as activated T cells[14]. In osteoblasts and under steady-state conditions, vitamin D, parathyroid hormone and prostaglandins lead in induced RANKL expression. The binding of RANK and RANKL leads in osteoclast differentiation[15,16]. More specifically, during normal bone remodeling, RANKL is produced by cells of the bone marrow- supporting tissue and osteoblasts. RANKL binds to RANK on pre- osteoclasts resulting in their maturation and activation. Nuclear-factor κB (NF-κB), which is of importance in inflammation response, also plays a central role in osteoclast activation. NF-κB performs both aforementioned functions through regulation from interleukin-6 (IL-6). Pro-inflammatory cytokines play an important role in bone remodeling as indicated by the presence of interleukin-1 (IL-1), IL-6 and tumor necrosis factor-α (TNF-α) receptors on pre-and mature osteoclasts[17]. OPG is produced by osteoblasts and has the ability to bind to RANKL and block its functions resulting in decreased bone resorption[17,18].

Bone remodeling is a strictly regulated process, largely modulated by the application of different mechanical stimuli or by metabolic stress on the bone[3].

More specifically, local mechanical stress leads in bone resorption as an initial response[19]. The nature of the mechanical stimulus is of importance in the regulation of bone remodeling since different types of mechanical stimuli result in different responses. For example, constant repetitive application of mechanical force inducing high stress levels or unusual load distribution result s in elevated bone synthesis and high bone mass. Furthermore, short pauses between long periods of mechanical loading have been shown to enhance bone strength and structure[20]. However, static load, slow rates of pressure rotation as well as “predictable” pressure application, lead in decreased bone synthesis, enhanced bone resorption and thus low bone mass[21,22].

Bone remodeling and mechanostimulation have been shown to roughly follow these rules: Bone synthesis is promoted by dynamic and not static loading application. Short-term load applications are sufficient for adaptive response initiation and lead in increased bone formation whereas long-term load applications result in decreased bone synthesis and enhanced resorption[23,24]. In addition, the repentance of the same mechanical stimulus results in decreased response due to signaling prediction[25]. The application of these rules is evident in the effects of space microgravity, osteoporosis or paralysis on bone tissues, where bone loss is observed[20,26], as well as in the effects of tennis at a professional level on bone tissues, where bone growth is observed[27].

Signals of mechanical nature induce in osteoblasts and osteocytes the production and secretion of different types of molecules, which modulate osteoblast differentiation and proliferation[3]. Such mechanical stimuli can include flow of fluids, strain of the substrate, membrane deformation or stimulation of integrins, vibration, altered gravity forces and compressive loading[3]. Bone remodeling functions, after the application of different mechanical stimuli, are locally regulated by cytokines and growth factors among other molecules. More specifically, IL-1β, TNF-α, prostaglandin E2 (PGE2)[26,28], IL-6, IL-8, RANKL, OPG[27,29-31], insulin-like growth factor (IGF), transforming growth factor β-1 (TGFβ-1) and fibroblast growth factor (FGF)[32,33] have been demonstrated to be induced after application of mechanical stimuli. Additionally, it has been shown that mechanical stimulation in osteoblasts results in increased mRNA levels of osteopontin, osteocalcin, platelet derived growth factor and collagen types I and III[34,35].

Although some of the molecules taking part in mechanotransduction are known, the mechanisms behind it have not been fully elucidated.

The stage of osteoblast differentiation is shown to be of importance in osteoblast proliferation, apoptosis and translation of mechanical cues[36]. Furthermore, it has been shown that undifferentiated mesenchymal stem cells seem to respond more successfully to load application than mesenchymal stem cells that have already started to differentiate[37].

A diversity of molecules have been considered to play the role of mechano-sensors in differentiated osteoblasts: mechanical stimulation has been shown to lead in enhanced sensitivity and elevated open cation channels number[38,39], increased communication through gap junctions between osteoblasts as well as increased integrin production in osteoblasts[39]. Actin cytoskeleton abnormalities have been shown to prevent mechanical signaling and therefore the integrin network has been considered as the main candidate for transduction of mechanical signals[39]. On the other hand, a considerable number of research groups argue that cytoskeletal components involved in mechanotransduction differ depending on different types of stress or the response under study[39].

Integrins comprise transmembrane receptors connecting the extracellular matrix to the cytoskeleton[40]. Under mechanical signal application, integrins form complexes with molecules of the cytoskeleton with the help of the Rho family of Ras-related GTPases[40]. Rho family members also induce multiple kinase cascades and particularly mitogen-activated protein kinase (MAPK) cascades[40]. Rho and other Ras-related GTPases have been shown to play a role in osteoblast response after application of mechanical pressure[41]. More specifically, it has been shown that the continuous application of mechanical forces leads in deregulation of Rab and Rho GTPases activity in osteoblast-like cells[41].

Recently, another molecule, Polycystin-1 (PC1), was suggested to provide a link between environmental mechanical signals and their transformation towards biochemical signals. It has been shown that PC1 not only functions as a mechanosensor but that also conveys mechanical signals through the calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway and thereby regulates osteoblast- specific gene transcription as well as osteoblast differentiation[42].

The primary cilium, a cellular sensory system, has also been demonstrated to be of importance in the transfer of mechanical signals as well as in mesenchymal stem cell differentiation. Additionally it was shown that the cilium modulates fluid flow mechanotransduction in human mesenchymal stem cells by maintaining fluid flow-induced osteogenic gene expression elevation and preventing fluid flow-induced increased proliferation[43].

Following the reception of mechanical cues, the signal conveying the mechanical conditions of the extracellular environment is carried towards the nucleus through MAPK kinases and more importantly through extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs)[44,45]. ERKs, which in human osteoblasts seem to be induced by growth factors, estrogen and fluoride among others[45], have been shown to play a significant role in osteoblast maturation and in osteoblast biology in general[45-49]. Furthermore, duration and strength of JNK/ERK signaling is indicated to be significant in gene expression[50].

Following ERK/JNK activation, the signal is transmitted to transcription factors that alter gene expression, like Jun and Fos family members[51]. In their turn, c-Jun and Fos family members interact to form activator protein-1 (AP-1) transcription factor, which has been shown to be of major importance in osteoblast differentiation[52] since it regulates the expression of collagen type I, osteocalcin, osteopontin and osteonectin[52].

Application of continuous mechanical pressure in osteoblast-like cells as well as osteoblasts resulted in increased production of AP-1 components through activation of MAPK cascades[41,53,54]. However, data on c-Jun expression after mechanical stimulation are inconclusive with some research groups arguing that human osteoblast-like cells after mechanical loading over-express c-Jun[53] whereas others have opposing results[55,56]. However, the above mentioned differences could be attributed to application of different stress type or usage of different cell system. Finally, different types of mechanical pressure applied on osteoblasts seem to result in different composition AP-1 and therefore regulate gene transcription accordingly depending on the extracellular signal applied[57].

Application of short-term mechanical pressure activates both JNK2 and ERK2, with following activation of downstream molecules, like c-Jun, which alter the expression of osteoblastic genes[54]. More specifically, it has been demonstrated that short-term continuous mechanical stimuli of physiological intensity in osteoblast-like cells activates JNK and ERK members resulting in enhanced AP-1 DNA binding activity on the human L/B/K ALP gene and thus osteoblast differentiation[54]. This is further evidenced by the observation that osteoblast-like cells receiving mechanical stimuli synthesized increased quantities of type 1 collagen and osteocalcin, markers of early osteoblast differentiation[58].

PGE2 production has been shown to be induced in osteoblast-like cells after mechanical stimulation[59] and in osteoblasts under the effect of physiological stress, growth factors, hormones, trauma or inflammatory cytokines and its production leads in cAMP-dependent IGF-1 induction in osteoblasts[3]. IGF-1 and IGF-2, in turn, induce osterix (Osx) transcription factor expression in osteoblasts[60], induce osteoblast function in vitro as well as lead in increased bone mass in vivo[61]. PGE2 is also shown to lead in increased Runx2 expression in vivo[62]. Downstream of PGE2, TGF-β expression, which leads in proliferation of osteoblasts and extracellular matrix synthesis[63], has been found increased in human osteoblast-like cells under mechanical stimulation. Furthermore, TGF-β receptor 1 comprises a Runx2 target in osteoblasts[64]. Those two observations combined explain why Runx2 knockout mice demonstrate characteristic abnormal extracellular matrix formation due to decreased number of mature osteoblasts[65,66].

Nitric oxide (NO) production in osteoblasts is another response to mechanical stimulation. NO functions through the MEK/ERK cascade by binding to a regulatory site on Ras leading in cell proliferation and extracellular matrix production[67]. Following, cyclooxygenase 1 (Cox1), Cox2, ERK1 and ERK2 are activated and result in bone matrix formation[68].

Additionally, signals of mechanical nature have been shown to promote vascular endothelial growth factor-, bone morphogenetic protein 2 (BMP-2)- and BMP-4- dependent and PGE2- independent increased expression of IGF-1[69]. BMPs result in bone synthesis in osteoblasts[70] and BMP-2 expression promotes Runx2, Osx and Dlx5 expression[71].

Mechanical cues also promote the expression of genes that encode for c-Fos, early growth response factor 1 (Egr-1) and basic fibroblast growth factor (bFGF) which have been shown to promote cell growth in MC3T3-E1 osteoblasts[22].

The nature the mechanical signal determines whether bone or cartilage formation will occur[72]. More specifically, application of pressure of high frequency and low intensity in bone cells in vitro, results in elevated extracellular matrix (ECM) disposition and thus increased bone formation[73]. On the contrary, mechanical loading of high intensity on osteoblasts leads in BMP extracellular antagonists expression and therefore results in inhibition of osteoblast development[74]. In addition, the application of continuous mechanical forces on osteoblastic cells in vitro promotes inflammatory cytokines and their receptors expression[75]. More specifically, IL-1b production is found elevated under such mechanical stimuli, and is accompanied by RANK-RANKL signaling pathway activation and thus bone resorption[76]. Stimuli from short periods of fluid flow or cyclic substrate tension at physiological intensity levels promote osteoblast proliferation and survival[77]. Mechanical signals of physiological intensity levels are associated with survival of human osteoblasts and several studies suggest that pro-survival proteins promote the production of survival factors like IGF-1 or IGF-2 and activate estrogen receptor[78]. It has also been shown that gravitational force maintains osteoblast survival whereas when gravitational force is not taking place, osteoblasts are led to apoptosis through reduced DNA binding of an important for survival transcriptional factor[18]. In vivo, the absence of mechanical signals promotes osteoblast apoptosis and thus osteoporosis[72]. The application of excessive mechanical force in vitro leads in cell detachment from their adhering surface[79] as well as in a form of programmed cell death called anoikis[80].

Mechanical stimulation in osteocytes has also been under investigation since it may lead in better mechanotransduction understanding and may represent a potent therapeutic target against bone degenerative diseases. Recent studies have underlined the role of osteocytes in bone remodeling since their absence in mice led in fragile bones, microfractures, deregulated osteoblast functions, bone loss in the trabeculae as well as adipose tissue proliferation in the marrow indicating an aging skeleton. In addition, these mice could not experience bone loss due to unloading, an event that indicates osteocytes’ importance in the procedure of mechanotransduction[81] (Figure 1).

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Figure 1 Mechanotransduction: Deregulation, associated disorders and therapeutic implications. Causes and effects of distorted mechanotransduction and the role of mechanical stimulation in the treatment of various pathophysiologies. PC1: Polycystin-1; IGF: Insulin-like growth factor; TGF-β: Transforming growth factor β; BMP: Bone morphogenetic protein. NFAT: Nuclear factor of activated T-cells; MAPK: Mitogen-activated protein kinase; Runx2: Runt-related transcription factor 2.

Runx2 which is known to play a significant role in osteoblast differentiation has been shown to be the recipient of mechanical signals in human osteoblast-like cells[82]. As it has been demonstrated, continuous mechanical stimuli of low intensity in human osteoblast-like cells of the periodontal ligament (PDL) result in elevated Runx2 expression and DNA- binding capacity. The mechanical signal, according to the researchers, initiates at the plasma membrane and more specifically from integrins and travels towards the nucleus through MAPK cascades. In the nucleus, the signal targets Runx2 and induces its expression[82]. More specifically, Runx2 demonstrates increased expression at both mRNA and protein levels as well as elevated DNA binding activity. During this process, ERK1 and ERK2 are activated in a parallel manner with the Runx2 DNA- binding capacity elevation. After their activation, ERKs interact, phosphorylate and activate Runx2 in vivo causing osteoblast maturation[7,82].

Runx2 expression depends on an autoregulatory mechanism[83]. More specifically, activated by mechanical stimuli ERKs phosphorylate and activate already existing Runx2 molecules. Those activated Runx2 molecules bind to Runx2 promoter inducing Runx2 expression[82]. In addition, a canonical AP-1 binding site has been found in Runx2 promoter which potentially plays a role in the regulation of Runx2 expression. AP-1 and Runx2 proteins have also been shown to interact and regulate collagenase-3 expression[84].

NF-κB transcription factor in mechanotransduction

NF-κB transcription factor which is implicated in inflammatory response signaling[31] also plays a crucial role in osteoclast formation and thus bone resorption[85]. NF-κB, which is activated either through the RANK-RANKL system or potentially through integrins that transmit signals of mechanical nature to src-kinases[86], besides its role in osteoclast maturation, may be implicated in osteoblast differentiation under mechanical stimulation. This is indicated by the fact that NF-κB is found to be activated and then translocated in the nucleus of osteoblasts that receive mechanical stimuli[26,87] where it has been hypothesized to promote the transcription of osteoblast-specific genes.