Mechanotransduction in osteoblast regulation and bone disease

Review

Mechanotransduction in osteoblast regulation and bone disease Katerina K. Papachroni1, Demetrios N. Karatzas1, Kostas A. Papavassiliou1, Efthimia K. Basdra2 and Athanasios G. Papavassiliou1 1

Department of Biological Chemistry, University of Athens Medical School, 11527 Athens, Greece Department of Histology and Embryology, Craniofacial Bone Tissue Unit, University of Athens Medical School, 11527 Athens, Greece

2

Osteoblasts are key components of the bone multicellular unit and have a seminal role in bone remodeling, which is an essential function for the maintenance of the structural integrity and metabolic capacity of the skeleton. The coordinated function of skeletal cells is regulated by several hormones, growth factors and mechanical cues that act via interconnected signaling networks, resulting in the activation of specific transcription factors and, in turn, their target genes. Bone cells are responsive to mechanical stimuli and this is of pivotal importance in developing biomechanical strategies for the treatment of osteodegenerative diseases. Here, we review the molecular pathways and players activated by mechanical stimulation during osteoblastic growth, differentiation and activity in health, and consider the role of mechanostimulatory approaches in treating various bone pathophysiologies. Osteoblasts: origin and organization Osteoblasts (see Glossary) originate from the non-hematopoietic part of bone marrow, which contains a group of fibroblast-like stem cells with osteogenic differentiation potential, known as the mesenchymal stem cells (MSCs) and also referred to as skeletal stem cells (SSCs), bone marrow stromal cells (BMSCs) and multipotent mesenchymal stromal cells (MMSCs) [1,2]. MSCs are capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes and, possibly, but still controversially, other non-mesoderm-type cells, for example, neuronal cells or hepatocytes [3,4]. Osteoblasts are key components of the bone multicellular unit (BMU), the basic skeletal anatomic structural unit [5]. BMU consists of bone-forming cells (osteoblasts, osteocytes and bone-lining cells), bone-resorbing cells (osteoclasts), the precursor cells of both, and their associated cells (e.g. endothelial cells and nerve cells) (Box 1). Bone architecture is formed or changed in any of three ways, namely, osteogenesis, bone modeling and bone remodeling. Osteogenesis occurs during the initial production of bone and is divided into two sub-types, intramembranous ossification and endochondral ossification. Once bone is formed, substantial changes in structure and shape are brought about by bone modeling, in which osteoblasts and osteoclasts act independently. Conversely, localized renewal of bone is brought about by bone remodeling, in Corresponding author: Basdra, E.K. ([email protected]).

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which the actions of osteoblasts and osteoclasts are coupled and bone resorption and formation occurs at the same spot on the bone surface. In humans, the osteoid part of each bone is renewed by remodeling approximately every two years throughout life (Box 1). Osteoblast growth and differentiation is determined by a complex array of growth factors and signaling pathways Growth factors Three families of growth factors influence the main aspects of osteoblast activity and induce, mediate or modulate the effects of other bone growth regulators. These are: (i) members of the transforming growth factor-b (TGF-b) family that promote osteoblast differentiation; (ii) the insulin-like growth factors (IGFs), which induce osteoblastogenesis via activation of osterix gene expression; and (iii) the bone morphogenetic proteins (BMPs), the autocrine and paracrine anabolic action of which is mediated by their specific receptors [6] (Box 2, Table 1). Because none of these growth factors are specific for cells in the osteoblastic lineage, mechanisms that promote skeletal tissue specificity are necessary, and these involve interactions with Glossary Bone multicellular unit (BMU): a local group of cells with finite lifetime that mediate bone remodeling. Each unit consists of bone-lining cells, osteoblasts, osteocytes, osteoclasts, their precursor cells, and their associated cells (endothelial and nerve cells) (Box 1). Bone remodeling: a dynamic, lifelong process of reshaping and replacing bone during growth and after injury. Chondrocyte: a polymorphic cell forming cartilage. These cells occupy lacunae distributed through the bone matrix. Mechanotransduction: a three-leg conversion of mechanical cues to electrical or biochemical signals, involving mechanosensing, signal transduction and effector-cell response (Box 2). Osteoblast: a cell originating from mesenchymal stem cells, responsible for the synthesis of bone matrix. Osteoclast: a multinucleated cell, differentiated from hematopoietic monocyte and macrophage precursors, which coordinates resorption of bone. Osteocyte: a terminally differentiated osteoblast trapped in its secreted matrix. Osteocytes sense mechanical signals and initiate events of bone remodeling. Osteopenia: a condition of bone in which there is a generalized reduction in bone mass; however, this is less severe than that in osteoporosis. Osteoporosis: skeletal abnormality characterized by decreased bone mass owing to the resorption of bone at a rate that exceeds bone synthesis. Periodontal ligament (PDL): a fibrous connective tissue surrounding the root of a tooth that separates it from and attaches it to the alveolar bone. It contains fibroblasts in undifferentiated mesenchymal form, which under appropriate conditions can generate multiple types of more differentiated, specialized cells that are involved in the homeostasis of both the ligament and adjacent bone tissue.

1471-4914/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2009.03.001 Available online 8 April 2009

Review Box 1. Bone multicellular unit (BMU)  Each unit is organized into a ‘cutting cone’ of osteoclasts which reabsorb bone, followed by a trail of osteoblasts reforming the bone; the end product is a new osteon.  Each BMU has a finite lifetime, so new units are continuously formed as old units die.  In normal bone, the number of BMUs, the bone resorption rate and the bone formation rate are all constant.  In adults, bone removed by a BMU is not completely restored and a small amount of bone is permanently lost; this is a normal phenomenon and accounts for the gradual loss of bone with aging.  Pathologic conditions in bone could increase the amount of irreversible bone loss by inducing either overactivity of osteoclasts or decreased capacity of osteoblasts to rebuild new bone  A permanent increase in bone mass does not normally occur in adults, except possibly in instances of periosteal bone formation owing to exercise  To understand or treat metabolic bone diseases, it is important to identify abnormalities in the BMU. This is best achieved by histomorphometric analyses of a bone biopsy after labeling of the bone with fluorescent calcium binding dyes (e.g. xylenol orange, calcein green); this technique permits quantification of rates of bone formation, resorption and number of BMUs.

other circulating hormones (including glucocorticoids, sex steroids, parathyroid hormone [PTH] or prostaglandin E2 [PGE2], which themselves have well-known effects on bone biology) in addition to the action of specific intracellular mediators on bone-specific transcription factors. Other growth factors, including vascular endothelial Box 2. Mechanotransduction The mechanical cues sensed by an osteocyte are converted into electrical or biochemical signals by mechanotransduction, which is brought about by three coupled procedures, namely mechanosensing (biochemical coupling), signal transduction and effector-cell response. The two most studied mechanosensors in osteoblast biology are integrins and mechanosensitive calcium channels, but the group also includes cell–cell adhesion elements (cadherins, gap junctions), surface processes (primary cilia, stereocilia), other membrane elements (caveolae, surface receptors), cytoskeleton constituents (microfilaments, microtubules, intermediate filaments), ECM particles (collagen, fibronectin, proteoglycans, basement membrane) and other cell–ECM adhesions (focal adhesions) [5], which act independently and interactively. Upon stimulation of the mechanosensors, and provided that intracellular structural integrity of cytoskeletal microfilaments exists, key intracellular enzymes are induced. MAPKs, Cox-2, NO, TNF-a and Wnt–b-catenin are activated in a duration- and type-ofapplied-force-dependent manner [15]. Mechanostimulation of osteoblasts also induces secretion of growth factors, including IGF, VEGF, PDGF, bFGF, TGF-b and the BMP, which are considered to be principal local regulators of osteogenesis, although none is specific for cells of the osteoblast lineage. Skeletal tissue specificity occurs through interactions among these growth factors with circulating hormones, or through specific intracellular mediators. Moreover, events that link these growth factors to nuclear proteins occur in response to glucocorticoids, sex steroids, PTH, or PGE2, which themselves have well known effects on bone biology [84]. The activation of intracellular signaling pathways and growth factors converge to activate transcription factors, namely Runx2, the principal transcriptional regulator of osteoblast differentiation, and Osterix, b-catenin and ATF4, which act downstream of Runx2, and other transcription factors that contribute to the control of osteoblastogenesis, including the activator protein-1 (AP-1), C/ enhancer-binding proteins (EBPs), PPARg and homeodomain, helix–loop–helix proteins [17].

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growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), are all secreted by the surrounding blood vessels and are involved in angiogenesis. VEGF promotes differentiation of osteoblasts and increases the mineralization of regenerated bone [7]. The role of PDGF is limited to situations associated with inflammation and repair, and FGF stimulates chondroblast and osteoblast replication but suppresses new bone matrix collagen synthesis [8] (Table 1). Notably, because some of the bone growth factors are secreted by peripheral tissues and their activity is regulated by specific endocrine factors, considerable variation in their expression or in the expression of their receptors on osteoblasts would be expected in response to intermittent or repeated exposure to normally controlled systemic hormones. Transcription factors Regulation of osteoblastic growth and differentiation occurs through osteogenic signaling pathways. A hierarchy of transcription factors is induced and is expressed in a defined temporal sequence to promote bone formation. Osteoblast differentiation starts with the commitment of osteoprogenitor cells from mesenchymal cells, followed by their progressive differentiation into mature functional osteoblasts expressing osteoblast phenotypic genes, and terminates with their transition into the osteocyte state within the bone matrix or with cell death for a fraction of the osteoblasts [9]. Runx2 is an essential bone-specific transcription factor. It is also essential for hypertrophic cartilage formation, which ultimately serves as a template for osteoblastic bone formation. Its expression is both necessary and sufficient for mesenchymal cell differentiation towards the osteoblast lineage. Runx2 is a member of the runx family, which comprises three genes, runx1, runx2 and runx3 (cbfa2, pebp2aB, AML1–3, respectively). All three genes encode proteins that harbor a DNA-binding domain and form heterodimers with the transcriptional co-activator core binding factor b (Cbfb), also known as polyoma enhancer-binding protein 2b (Pebp2b), in vitro [10]. Runx2 was discovered in searches for osteoblast-specific transcription factors by studying the regulation of expression of osteocalcin, the only gene expressed exclusively in osteoblasts and in no other extracellular matrix (ECM)-producing cell type. Runx2 acts as a master regulator that binds to osteoblast-specific cis-element 2 (OSE2) found in the promoter region of all major osteoblast-related genes. It is expressed very early in skeletal development, first appearing in the nascent mesenchymal cells in areas destined to become bone and persists in post-natal life through subsequent stages of bone formation [11]. Ectopic expression of Runx2 in mesenchymal cell lines leads to upregulation of osteoblast-specific genes, including osteocalcin, alkaline phosphatase, collagenase-3 (matrix metalloproteinase-13 [MMP-13]), bone sialoprotein (BSP) and collagen type I a 1 (COLIA1) [12]. Gene inactivation in transgenic mice (runx2 / ) leads to complete lack of intramembranous and endochondral ossification owing to lack of mature osteoblasts [13], but the mesenchymal cells in these organisms retain the potential to differentiate into adipocytes and cartilage-forming chondrocytes, 209

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Table 1. Involvement of growth factors in osteoblast differentiation Growth Origin of secretion factor  Secreted by the liver as a IGFs result of stimulation by the growth factor (GH)

TGF-b family



BMPs

 

VEGF



PDGF



Effects on osteoblasts

 IGF-1 triggers osteoblast proliferation, increases bone collagen synthesis and decreases collagen degradation  IGF-I and -II promote Osx expression in osteoblastic cells, trigger osteoblast induction in vitro and a transient increase in bone mass in vivo Secreted by many organs,  Stimulate the production and deposition of ECM proteins and has an autocrine action  Potent inducers of committed bone on osteoblasts cell replication and osteoblast matrix production  TGF-b induces Runx2 in vivo Members of the TGF family  Autocrine and paracrine action Concentrated in the organic mediated by their kinase receptors matrix of bone, released after  Induce early precursor bone cell a fracture or during bone replication and osteoblast commitment resorption  BMP-2 promotes Runx2 expression in mesenchymal osteoprogenitors and osteoblastic cells and Osx expression in osteoblastic cells  Regulates vascularization of Expressed in several tissues, developing bones and osteoclast exerts its role in endochondral activity, involved in bone repair ossification through its expression and release by  Key component of a chondrocyte hypertrophic chondrocytes survival pathway,controls osteoblastic activity Synthesized by megakaryocytes,  Potent mitogen and chemoattractants transported in blood by platelets for target cells such as diploid fibroblasts and osteoblasts

thus underlining the role of Runx2 in the specific commitment of cells in the osteoblast lineage. Heterozygous mice (runx2 /+) demonstrate specific skeletal abnormalities that are characteristic of the human heritable skeletal disorder cleidocranial dysplasia (CCD) [14]. Interestingly, although Runx2 is essential for bone formation, its tissue-specific overexpression in transgenic mice leads to osteopenia, with decreased bone mineral density and multiple fractures, thus indicating that Runx2 negatively controls osteoblast terminal differentiation and maintains osteoblastic cells in an immature state [15]. Overall, Runx2 has a dual biological role in the osteogenic lineage. It promotes attenuation of osteoblast growth and phenotypic maturation [16], while simultaneously interacting with several regulatory proteins within the nuclear architecture to activate or repress genes that control the program of osteoblast proliferation and differentiation [11]. Given its pertinent role in the osteoblastogenic process, Runx2 expression and activity are themselves tightly controlled by several transcription factors, protein–DNA or protein–protein interactions, which ensure prompt and accurate activation of the osteoblastogenic process [17]. The transcription factors include Osterix (Osx), b-catenin, ATF4 and distal-less homeobox 5 (Dlx5), which have important roles in osteoblastogenesis and which all cooperate with Runx2 to promote cell differentiation down the osteoblast lineage. Osx is a zinc-finger transcription factor specifically expressed by osteoblasts [18]. Gene inactivation (osx / ) 210

Effect of mechanostimulation on Refs growth factor levels  Stretching (cyclic and static) of osteoblasts [6,67] in vitro [MC3T3-E1] increases IGF-mediated signaling and promotes late osteoblast and early osteocyte differentiation

[39,40,81]  In human osteoblast-like cell line, SaOS-2 application of mechanical strains within physiological range (2000 me) has high inductive effect, hyperphysiological range (200 000–300 000 me) decreases bone-forming indices  Compressive force of human SaOS induces [6,37,49] BMP-mediated upregulation of Runx2 and Smad1 phosphorylation  In vitro sinusoidal cyclic stretch on cultured human spinal ligament cells augments BMPs mRNA

 Increased mechanical load in PDL and stretch deformation in rat and human osteoblasts increases VEGF expression

[7,82]

 Fluid shear stress in SaOS cells increases PDGF levels via cation channel functions

[8,83]

results in embryos with both hypertrophic cartilage and normal levels of Runx2, thereby suggesting that this factor operates downstream of Runx2. Consistent with this, osx transcription is positively regulated by Runx2 and although Osx has been shown to form a complex with the nuclear factor of activated T cells (NFAT), resulting in activation of COLIA1 promoter activity, its mode of action remains elusive [19]. Other studies have shown that constitutive activation of NFAT activates the Wnt signaling pathway and results in augmentation of bone formation and bone mass [20]. Additionally, p53 has a negative effect on osx transcription and thus downregulates osteoblastogenesis [21]. The activation of the Wnt–b-catenin signaling pathway has an anabolic osteoblastic role. It involves the accumulation of b-catenin and its translocation to the nucleus, where it binds to T-cell factor (TCF) or lymphoid enhancer factor (LEF) transcription factors and activates downstream genes and pathways, including runx2. Inactivation of b-catenin blunts osteoblast differentiation from mesenchymal progenitors, indicating that b-catenin has an essential role in osteoblast differentiation in vivo [22]. ATF4, also known as cyclic adenosine monophosphate (cAMP)-response-element-binding protein 2 (CREB2), interacts with Runx2 to regulate the transcriptional activity of osteocalcin [23]. ATF4 can be phosphorylated by the kinase Rsk2, and thus controls amino acid transportation in osteoblasts, an important step in bone formation. The biological importance of this factor was revealed by human genetic studies because RSK2 is the

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Figure 1. Schematic diagram of interactions of different signaling pathways during mechanical stretching. Membrane-bound receptors such as Ca2+ channels, integrins, Gproteins, IGF and TGF-b and/or BMP receptors are stimulated by mechanical forces, resulting in induction of several transcription factors that regulate osteoblast differentiation and formation. AP-1 and Runx2 are induced mainly through MAPK and SMADs pathways. Runx2 is also stimulated via the Wnt pathway, involving b-catenin and TCF or LEF factors. PLC–PKA pathway contributes to NF-kB, Cox-2 and CREB induction. Abbreviations: AP-1, activator protein-1; b-cat, b-catenin; DAG, diacylglycerol; FAK, focal adhesion kinase; G, G-protein; IP3, inositol (1,4,5)-trisphosphate; MEKK, MAPK kinase kinase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PYK2, proline-rich tyrosine kinase 2.

gene that is mutated in Coffin-Lowry syndrome, an X-linked mental retardation condition associated with skeletal abnormalities, largely caused by lack of ATF activation [24]. ATF4 inactivation in mice causes delayed skeletal development that progresses to severe low-bonemass phenotype [25]. Moreover, ATF4 is required for proper synthesis of type I collagen, although this function is mediated by its capacity for efficient amino acid import into osteoblasts [25]. Dlx5 belongs to the family of homeobox proteins (Msx1, Msx2, Dlx5, Dlx6) that act as repressors or activators of transcription and are essential for normal ossification [26]. Dlx5 is known to activate the expression of runx2 and bone markers BSP and osteocalcin [27]. Moreover, dlx5 / osteoblasts in culture display reduced proliferation and differentiation rates and reduction of runx2, osx, osteocalcin and bone sialoprotein expression. Femurs of the dlx5 / transgenic mice exhibit considerable increases in osteoclast number and given that dlx5 is not expressed by osteoclasts, it could be that its osteoblastic expression might control osteoblast and osteoclast coupling. These findings suggest that Dlx5 is a central regulator of bone turnover because it induces direct bone formation and indirect bone resorption [28]. Nuclear factor-kB (NF-kB) is postulated to activate transcription of osteoblast-specific genes. Its activation is

mediated by src-kinases, perhaps through the mechanosensing action of integrins [29]. However, NF-kB also has a crucial role in osteoclast formation and bone resorption [30]. Indeed, in a mouse model of bone loss arising from a lack of estrogens, NF-kB inhibitors substantially hindered the induced bone destruction by preventing the increase in osteoclastic bone resorption [31]. NF-kB acts through the receptor activator of NF-kB ligand (RANKL) pathway and promotes differentiation of osteoclasts [32]. Diverse mechanical signals trigger osteoblast differentiation Osteoblast differentiation is triggered by mechanical stimulation, which induces the secretion of hormones and growth factors, thus affecting the differentiation and proliferation potential of osteoblasts (Box 2, Table 1 and Figure 1). Osteocytes are the mechanotransducer cells of the bone. They orchestrate the combined action of hormones and growth factors and promote osteoblastogenic events in response to a range of mechanical stimuli, including (oscillating) fluid flow, substrate strain, membrane deformation or integrin stimulation, vibration, altered gravity and compressive loading (increased hydrostatic pressure). Fluid flow strain, generated upon induced movement of extracellular fluid through the bone 211

Review lacuno-canalicular system after mechanical loading, stimulates bone cells through streaming potentials (which originate when electrolytes are driven by a pressure gradient through a channel or porous plug) and wall shear stress [33]. Substrate strain results from application of mechanical force to cells adhered on elastic substrata, either in single bouts (static strain) or in repeated cycles of positive or negative strain (cyclic strain). A detailed presentation of the molecular outcomes of mechanical stimulation of human osteoblast cell lines has been provided by Scott et al. [34]. Release of PGE2 is a prominent load-induced response of osteoblast-like cells [35]. PGE2 is produced by osteoblasts in response to physiological stress, growth factors, hormones, trauma or inflammatory cytokines and induces cAMP-dependent IGF-I expression by osteoblasts (Box 2). IGF-I and IGF-II promote Osx expression in osteoblastic cells [36] and trigger osteoblast induction in vitro and a transient increase in bone mass in vivo [37]. The in vivo anabolic effects of PGE2 are also associated with increased expression of Runx2 [38]. Downstream of PGE2, anabolic TGF-b mRNA and protein levels are both elevated in human osteoblast-like cells. The multifunctional mitogenic and matrix-inducing effects of TGF-b in bone [39] and the identification of TGF-b receptor1 (TGF-bR1) as a crucial target gene for Runx2 in osteoblasts [40] might explain, in part, why mice lacking Runx2 have diminished numbers of osteoblasts and defects in ECM deposition [13]. Nitric oxide (NO) secretion is another key load-sensing event. Animal studies with flow-stimulated or cyclically stretched primary osteoblasts have demonstrated secretion of NO, which binds to a regulatory site on Ras and potentially stimulates proliferation and ECM production through the Ras–Raf–mitogen-activated protein kinase (MEK)– extracellular signal-regulated kinase (ERK) cascade [41]. Cyclo-oxygenase (Cox) 1,2 activation downstream of NO and ERK1,2 activation is also necessary for induction of anabolic functional changes in osteoblasts [42]. Mechanical stimulation also leads to upregulation of IGF-I (Box 2). This upregulation is reliant on the presence of VEGF, BMP-2 and BMP-4, but is independent of PGE2 [43]. The induced growth factors activate phosphoinositide 3-kinase (PI3-K)–protein kinase B (Akt), MAPKs and SMAD signal transduction pathways [44]. BMPs act anabolically on osteoblasts through autocrine and paracrine mechanisms via their receptors, which can be tyrosine kinases and serine/threonine kinases [44]. The BMP-2-induced signaling pathway leads to the expression of three of the osteogenic transcription factors Runx2, Osx and Dlx5 [45]. Mechanical strain also induces upregulation of the growth-related genes c-fos, early growth response factor 1 (egr-1) and autocrine basic fibroblast growth factor (bFGF) and induces growth in MC3T3-E1 osteoblasts. Specifically, 15-min pulses of gravity stress on osteoblasts, analogous to in vivo physiological levels such as those seen in walking or running, increase osteoblast proliferation after 24 h [46]. Differences in mechanical loading conditions can direct bone versus cartilage formation [47] and stretch induces differentiation in periodontal ligament (PDL) osteoblastlike cells [48]. Individual bone cells increase deposition of 212

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Figure 2. Molecular interactions between osteoblasts and osteoclasts during bone remodeling. Application of mechanical stretch on osteoblasts induces augmentation of OPG and reduction of RANKL expression, which in turn reduces RANKL–RANK interaction and osteoclast differentiation. MAPK p38 mediates OPG induction, whereas MAPK ERK mediates the activation of transcription factors that stimulate RANKL expression. Although OPG and RANKL are controlled by as yet unknown molecules, osteoclast maturation is driven by RANK, which potentiates c-Fos and NF-kB, stimulates NFATc1 gene and leads to differentiation of osteoclasts.

bone ECM when exposed to mechanical stresses with high frequency and low strain in vitro, just as they do within whole bone [49]. However, excessive compressive forces on osteoblasts induce expression of extracellular antagonists of BMPs and thus inhibit osteoblastogenesis [50]. Moreover, continuous compressive force in osteoblastic cells in vitro induces production of inflammatory cytokines and their receptors in osteoblasts [51]. This phenomenon is enhanced by the autocrine action of interleukin (IL)-1b, the levels of which are increased by mechanical stress, and can be linked to the signaling pathway of the RANKL and its corresponding receptor (RANK). The RANKL–RANK system is responsible for inducing osteoclastogenesis (Figure 2). RANKL, a tumor necrosis factor (TNF) superfamily member, is a cell-surface molecule expressed by a large set of different cell types, including activated T cells. Under steady-state conditions, its expression is induced in cells of the osteoblastic lineage in response to osteotropic factors, such as vitamin D, parathyroid hormone and prostaglandins. The interaction of RANKL with its receptor RANK is modulated by osteoprotegerin (OPG), a secreted glycoprotein, which was identified as a soluble factor that strongly suppresses osteoclast differentiation both in vitro and in vivo. OPG can suppress bone resorption and increase bone mass by binding via its TNF receptor (TNFR) domains to TNF domains of its natural ligand, RANKL [52]. Short periods of fluid flow or cyclic substrate strains at physiological levels act favorably to promote the proliferation and survival of osteoblasts [53]. Physiological strain

Review induces the expression of survival signals in human osteoblasts and, although often inconsistent, some studies suggest that the expression of pro-survival proteins, including clustering and upregulation of b-integrins [6], causes release of autocrine and paracrine survival factors such as IGF-I or IGF-II and activation of the estrogen receptor [54]. Appropriate exercise can enhance bone density, which can be tracked in vivo, as can its decline after cessation of exercise [55] (Box 1). Moreover, gravitational force ensures the survival-promoting behaviour of osteoblasts. Its absence causes a cytokine-mediated reduction of DNA binding of a survival-promoting transcription factor and disrupts mitochondrial function (i.e. alters Bcl to Bax ratio), thereby sensitizing osteoblasts to apoptosis [56]. Interestingly, the absence of any mechanical stimulation in vivo leads to apoptosis of osteoblasts and osteoporosis [47], whereas inappropriately high strain also results in considerable cell detachment and rupture of cell adhesions, which are necessary for physiological resistance to high levels of strain [57]. Detachment of anchoragedependent cells from the surrounding extracellular matrix leads to a form of programmed cell death known as anoikis [58], but there is no evidence that this phenomenon occurs in vivo in response to high loads. In summary, it is clear from the aforementioned studies that bone cells are very sensitive to the magnitude of mechanical strain, with too much or too little mechanical strain causing cell death. Model systems for the study of mechanotransduction in osteoblasts Systems of osteoblast-like cells have been used in vitro to model mechanotransduction. These cells originate either from healthy tissue (e.g. human PDL or mouse MC3T3-E1 calvaria cells) or from osteosarcomas (e.g. MG-63, SaOs cells), which acquire osteoblast-like characteristics. The magnitude of the mechanical stimuli applied to the cells is expressed as an absolute number or a percentage, usually expressed in microstrains (me). Physiological levels of strain of >5000-me range can cause fractures or breaks in human bone, depending on the condition of the bone [44]. Cells in culture are mechanostimulated using fluid flow, four-point bending and substrate stretch, although some studies have used gravity force, vibration, magnetic bead twisting, atomic force or shockwaves [34]. Osteoblast response depends on the type, duration and level of stress and all the methods of introducing strain result in the osteoblast being mechanically stimulated. A comprehensive review on the comparison of mechanical strain techniques and the likely mediating pathways has been provided by Hughes-Fulford [44] and a collection of in vitro experimental data of cellular physiology upon mechanotransduction in bones are summarized by Scott et al. [34]. An excellent model system for the study of the mechanical-strain-induced molecular changes in the osteoblast is the model of PDL cells [59]. The cells in the PDL are predominately fibroblasts, in an undifferentiated mesenchymal form and comprise a cell-renewal system in a steady state [60]. They can be readily and non-invasively isolated from healthy extracted molars and pre-molars, and have been used successfully for guided tissue regeneration (GTR) on biological membrane substrates with comparable results

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to osteoblast-like cells [61]. Under appropriate induction conditions, PDL cells can generate multiple types of more differentiated, specialized cells that are involved in the homeostasis of both the ligament and adjacent bone tissue [62]. Recently, highly purified periodontal stem cells (PSCs) and osteoprogenitors were isolated from adult human PDL capable of bone formation in vitro, thus further establishing their potential value for stem-cell-based bone tissue engineering in vivo [63]. PDL osteoblast-like cells posses all the osteoblastic properties, including the ability to secrete molecules that affect the osteoblast–osteoclast balance. Interestingly, studies of co-cultures of PDL osteoblast-like cells and osteoclasts could shed light on the molecular mechanisms underlying their interactions. The role of mechanotransduction in bone disease The conversion of physical force into biochemical information is fundamental to development and physiology and goes beyond the skeletal system. In the vascular system, pressure and shear stress from pumping blood influence the morphology and pathology of the heart and vasculature, whereas hearing and touch are based on neural responses to pressure, to mention but a few systems. Bone is specifically designed to respond to and adapt to changes in mechanical loads. The mechanisms by which increases (overloading) or decreases (underuse) in mechanical loading cause bone formation or resorption are the same, although the direction of the changes is different. It is worth noting that overloading and underuse should be defined as the increase and the decrease, respectively, in activity relative to that in which the skeleton is currently habituated or adapted, and thus there are no absolute levels of activity that constitute overuse or underuse [47]. Some bone disorders are due to impaired skeletal mineralization (rickets and osteomalacia) or to adverse immunological reactions (osteoarthritis and rheumatoid arthritis). However, bone diseases almost invariably result from deviations from the dynamic balance of osteoclastic bone resorption to osteoblastic bone formation for each organism (Box 1). This divergence largely originates either from genetic abnormalities (e.g. mutation in the runx2 gene results in the human heritable skeletal disorder CCD [20]) or from aberrant mechanotransduction (ankylosing spondylitis, carpal tunnel syndrome, chronic back pain, disc degeneration). One of many manifestations of aberrant mechanotransduction ‘cross-talk’ between osteoblasts and osteoclasts is osteoporosis, in which an increased rate of bone resorption and reduced bone formation per se is observed (also see section on mechanostimulation as a therapeutic regimen for bone disease). Osteoporosis can originate from disease, or dietary or hormonal deficiency and manifests clinically with loss of bone density, thinning of bone tissue and increased vulnerability to fractures. Similar bone loss can result from decreases in mechanical loading owing to extended bed rest or exposure to microgravity [56]. Mechanostimulation as a therapeutic regimen for bone disease The rationale behind therapies that combat bone weakening and bone loss is to achieve a higher rate of bone 213

Review formation than resorption, either absolutely, as with anabolic treatments, or in relative terms, as with anti-resorptive agents. Either action translates clinically into increased bone mineral content, stabilization of bone architecture and reduced fracture risk, which is the ultimate clinical goal. Anabolic treatments involve intermittent PTH treatment, targeting the Wnt–b-catenin signaling pathway and administration of statins [64]. Anti-resorptive remedies are based on achieving impaired or diminished osteoclast function by interfering with RANKL or with downstream effectors of its signaling pathway in the osteoclasts [65]. Other current pharmacological treatments include administration of estrogen and selective estrogen receptor modulator (SERM) or bisphoshonates (BPs). The bisphoshonate family of drugs is currently the most widely used treatment for osteoporosis, tumor osteolysis, humoral hypercalcemia, multiple myeloma and Paget’s bone disease. BPs act by binding to mineralized bone surfaces in a high-affinity manner and, thus, inhibit resorption [64]. Mechanical stimulation can also reverse the process of bone loss, both by downregulating osteoclastogenesis and RANKL and by inducing bone formation. In vitro experiments in PDL and osteoblast cell lines have shown that OPG expression is increased upon application of tensile or cyclic strain, via activation of the p38 MAPK pathway [66,67]. Increased OPG levels prevent RANKL–RANK interaction and, thus, osteoclastogenesis (Figure 2). Moreover, RANKL itself is downregulated upon mechanical stimulation, via synthesis of Cox enzymes or prostaglandins and, thus, osteoclast formation is ablated [53,68]. Mechanical stimulation also interferes with Wnt signaling, as demonstrated by experiments in which dynamic strain applied on osteoblastic cells activates the Wnt–b-catenin pathway and thus induces osteoblastogenesis [69]. Mechanical strain is used for the treatment of various skeletal disorders. Considerable gain in bone mineral is obtained by strengthening exercises in individuals with established osteoporosis [70], and by a new physical therapy that combines the use of a device called spinal weighted kypho-orthosis (WKO) with specific back extension exercises – this has beneficial effects in reducing back pain, improving posture and reducing the risk of falls in women with osteoporosis who also have curvature of the spine [71]. Moreover, physical exercise acts proactively in preventing post-menopausal and age-related loss of bone mineral [72]. In degenerative disc disease (DDD) and other skeletal deformations, applied therapeutic approaches use dynamic stabilization implants and aim to maintain a functional mechanical environment. These restore the physiological motion, load distribution and intradiscal pressure and provide a suitable environment for further treatment options [73]. Bone fracture healing has also benefited from mechanostimulation of osteoblasts. The effects of loading depend heavily on the rate [74], mode [75] and magnitude of loading, and on the gap size of the fracture [76]. Osteoblasts and osteocytes respond differently in varied mechanical environments and, as such, rigid fixation of fractures results in direct bone formation and osteonal 214

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Box 3. Outstanding questions  What are the mechanisms by which bone cells can discriminate between mechanical stimuli of different magnitude, direction and duration?  How are RANKL and OPG controlled transcriptionally after mechanical stimuli?  Is it feasible to achieve ‘directed’ combinatorial therapies of mechanical stimuli and pharmacological compounds?

bridging of the fracture gap, whereas flexible fixation can result in indirect healing characterized by periosteal callus formation and enchondral bone formation [75]. Application of cyclic compressive displacements can enhance healing through increased callus formation and more rapid ossification [76,77]. Other forms of mechanical stimulation such as low-level vibrations at a level far below that which could cause damage to the bone represent a unique, non-pharmacological prophylaxis for osteoporosis. This was shown by studies in children with musculoskeletal disorders, young women with low bone mass and post-menopausal osteoporotic women, indicating that such stimulation can be efficacious in reversing and/or preventing bone loss [78]. Moreover, low-intensity pulsed ultrasound (LIPUS) has proven beneficial for the treatment of pathological and trauma fractures of bone because it promotes osteogenesis and therefore facilitates bone regeneration [79]. Finally, experiments using an extremely low-frequency pulsed electromagnetic field (PEMF) on osteoblasts have revealed that it promotes proliferation and osteoblast maturation of primary osteoblast cells [80]. Concluding remarks The adaptation of the skeletal system and its responsiveness to different mechanical and hormonal environments makes skeletal cells valuable centers of information sorting, and highlights their capacity to respond to a wide range of stimuli. The complexity of the mechanisms for osteoblast differentiation and their interactions with osteocytes and osteoclasts demonstrate that the pathways presently known are probably incomplete and more molecules are yet to be identified. The mechanisms by which osteocytes sense the different characteristics of a mechanical stimulus and transduce them to osteoblasts remain elusive, as do the mechanisms by which osteoblasts consequently co-ordinate the activation of specific intracellular signaling pathways. Further investigation of the molecular basis of mechanotransduction in bone physiology and disease is needed before the therapeutic value of mechanically oriented strategies can be appreciated (Box 3). Such approaches provide a non-invasive means of bone strengthening in osteodegenerative diseases, thus obviating the side-effects associated with use of pharmaceuticals. However, it will be interesting to investigate whether or not approaches using targeted combinatorial therapies of mechanical stimuli and pharmacological compounds might be feasible. References 1 Abdallah, B.M. and Kassem, M. (2008) Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther. 15, 109–116

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