**4. Mechanosensation and mechanotransduction**

#### **4.1. Strain amplification**

Radiation pressure is a universal phenomenon in any wave motion involving sound. It is exerted on surface or media interfaces and acts in the direction of propagation of the wave thereby producing direct and indirect mechanical stress. Direct mechanical stress is produced by strain. Following mechanical deformation, bone exhibits electrical activity and cellular activation. It is unclear, however, whether the main responsible for bone electrical activity is piezoelectricity, streaming potentials, or ion channels and ATP receptor activities (see 3.4.1)

Indirect mechanical stress is produced by acoustic streaming and modal conversion. When acoustic waves are refracted from water to soft tissues, waves propagate longitudinally (in the same direction of the beam source) due to impedance similarity. Differently, when acoustic waves are refracted to materials with impedance mismatch, such as bones, modal conversion occurs, that is, shear waves (waves at right angles to the direction of the beam source) are produced along with longitudinal waves. Shear waves may produce direct mechanical

Acoustic radiation pressure decreases with the distance of the wave from its source; hence, radiation pressure gradients are formed within the fluid. As a result, fluid flow originates, which is called acoustic streaming. The flow is directed away from the transducer with gradual build up of the axial streaming speed with distance from the transducer and a peak of velocity in the focal region. The fluid flow continues beyond the focal region and returns to the transducer as recirculation vortices. Fluid flow can also build up again in the acoustic beam after a membrane. Acoustic streaming and microstreaming are often used as synonyms in the literature. Although both produce fluid flow which can modulate osteogenesis, they are distinct phenomena. As described above, acoustic streaming results from radiation pressure gradients, whereas microsteaming is generated by stable cavitation bubbles. Furthermore, not only mechanical deformation but also acoustic streaming increases cell membrane permea‐ bility and generates streaming potentials. It has not been shown, however, whether acoustic streaming directly affects cell membrane permeability, or triggers cellular reactions that increase membrane permeability [30, 37, 45, 48]. For a detailed explanation of streaming

Bone exhibits electrical activity when subjected to mechanical forces. The opposite is also true: bone undergoes deformation when exposed to electric potentials. For instance, ESWT induces transient cell membrane hyperpolarization. There are three possible contributors to electric potentials on bone. First, mechanically induced activity of ion channels and ATP receptors promotes ion transport between the intra and extracellular environments resulting in mem‐ brane action potentials; second, piezoelectricity, which is the generation of electricity when asymmetric crystalline materials—as those that form the extracellular matrix of bone—are subjected to strain; and finally, streaming potentials that result from mechanically induced

deformation to tissue, called shear stress [7, 27, 36, 45].

flow of fluid containing high conductivity ions [3, 4, 44, 49].

[4, 28, 34, 45].

36 Advanced Techniques in Bone Regeneration

potentials, see reference [3].

*3.4.1. Electric potentials*

The various cell types that populate bone—osteoblasts, osteocytes, osteoclasts, periosteal cells (fibroblasts and progenitor cells) and bone marrow cells (include mesenchymal stem cells) are responsive to mechanical stimulation. Bone is a hard material that can handle up to 2% of strain (i.e. 20,000 μstrain) without failure (fracture). However, based on in vitro studies, bone cells need strains up to 10% in order to direct their response to osteogenesis. In addition, a large amount of energy is lost during wave propagation within bone by means of attenuation and reflection; as such, bone cells may be exposed to low pressure waves. A possible explanation would be that strains are amplified at tissue level, so that cells are exposed to higher strain intensities. At the moment, that hypothesis could not be proved. Nevertheless, the following mathematic-based model supports that explanation [1–3, 41, 43, 44, 50–57].

The model for strain amplification was based on the microanatomy of osteocytes, which are the main mechanosensors of bone. Cytoplasmic processes of osteocytes are separated from their canalicular wall by a pericellular space filled with albumin-rich fluid. Moreover, cytoplasmic processes are anchored to their canalicular wall by transverse fibrils. When mechanically induced fluid flow collides with fibrils, hoop strains are generated on the membrane-cytoskeleton system of cytoplasmic processes. Hoop strains produce forces which are 20–100 times higher than at bone's surface. The magnitude of hoop strains depends on the relationship between fluid and transverse fibrils within pericellular space, and between cell membrane and cytoskeleton constituents (i.e. actin filaments and fimbrins) [3, 57–61].

#### **4.2. Mechanoreceptors**

Several structures at cell membrane act as "mechanoreceptors." Mechanically-induced structural deformation of mechanoreceptors triggers their activation. Sequentially, a cascade of biological reactions initiates and results in osteogenesis. Known mechanoreceptors of bone cells include integrins, ATP receptors, ion channels, growth factors (includes hormones) receptors, low-density lipoprotein receptors, frizzled proteins, G proteins and connexins. Among those mechanoreceptors, only integrins were proved to have a role in mechanosensa‐ tion of ESWT. Regarding mechanosensation of LIPUS, integrins, ATP receptors, growth factors receptors, low-density lipoprotein receptors and frizzled proteins have established participation. In the following sections, the molecular events triggered by LIPUS and ESWT are described. To date, RPWT effects on bone cells have not been investigated properly. It should be noted that acoustic loading refers only to LIPUS, ESWT or RPWT, and mechanical loading refers to any type of mechanical forces that may, or not, be acoustic loads. **Tables 2** and **3** enlist molecular events related to LIPUS and ESWT [1, 10, 50, 55, 62–66].

#### *4.2.1. Integrins*

Mechanoreceptors convert mechanical deformations into biological reactions, a process called mechanotransduction. Among mechanoreceptors, it is believed that integrins are vital for mechanotransduction. Evidence suggests the activation of all others mechanoreceptors and a multitude of signalling pathways are integrin-dependent. Therefore, osteogenic response of bone cells (adhesion, migration, differentiation and proliferation) depends on integrins. The expression of α2, α5, β1, β3 integrins subunits are increased by mechanical loading. Further‐ more, clusters of α5β1 and αvβ3 integrins formed at the deformation site—also known as focal adhesions—attract a number of cytoplasmic proteins and trigger a cascade of reactions [3, 10, 37, 43, 50, 53, 55, 60, 67–71].


**Table 2.** Signalling pathways triggered by acoustic stimulation.



**Table 3.** Biological effects of LIPUS and ESWT.

*4.2.1. Integrins*

37, 43, 50, 53, 55, 60, 67–71].

38 Advanced Techniques in Bone Regeneration

P2Y1 receptor activation

Mechanoreceptors convert mechanical deformations into biological reactions, a process called mechanotransduction. Among mechanoreceptors, it is believed that integrins are vital for mechanotransduction. Evidence suggests the activation of all others mechanoreceptors and a multitude of signalling pathways are integrin-dependent. Therefore, osteogenic response of bone cells (adhesion, migration, differentiation and proliferation) depends on integrins. The expression of α2, α5, β1, β3 integrins subunits are increased by mechanical loading. Further‐ more, clusters of α5β1 and αvβ3 integrins formed at the deformation site—also known as focal adhesions—attract a number of cytoplasmic proteins and trigger a cascade of reactions [3, 10,

**Signalling pathways LIPUS ESWT**

α5β1 integrin/FAK/Scr/Grb2/Sos/Ras/Raf/MEK/ERK X

α5β1 integrin/β-catenin X X

Ras/Rac1/NADPH/superoxide/ERK/cbfa1 X Ras/Rac1/NADPH/superoxide/HIF-1α/VEGF X

**Biological effects LIPUS ESWT** Increased expression of α5 and β1 integrins X X

α5β1-mediated FAK activation X X

Bax expression X X

α5β1 and αvβ3 integrins/FAK/Scr/Grb2/Sos/Ras/Raf-1/MEK/ERK/IKKα,β/IκBα/NFκB/cox-2/PGE2 X α5β1 and αvβ3 integrins/FAK/Scr/Grb2/Sos/Ras/Raf-1/MEK/ERK/IKKα,β/IκBα/NFκB/iNOS/NO X

α5β1 and αvβ3 integrins/FAK/PI3K/Akt/NFκB/cox-2/PGE2 X

AT1/ERK-1,2 X

α5β1 and αvβ3 integrins/FAK/PI3K/Akt/Bcl-2 X

Increased expression of α2 and β3 integrins X β1 and β3 integrins clustering X

αvβ3-mediated FAK activation X Increased IRS-1 activity X Increased P2X7receptor activation and activation, and ATP release X

mTOR activation X

ILK phosphorylation X

**Table 2.** Signalling pathways triggered by acoustic stimulation.

#### *4.2.2. ATP receptors*

ATP receptors promote the exchange of calcium from intracellular deposits to extracellular environment, or from extracellular environment to intracellular environment. ATP receptors complex with integrins and G proteins, and some (P2X7 and P2Y1) are activated by mechanical loading. By means that need to be explored, mechanically induced activation of P2X7 and P2Y1 induce osteogenic differentiation—represented by increased expression of cbfa-1, osterix, type I collagen, bone sialoprotein, osteopontin and osteocalcin—and osteoblasts proliferation [3, 64, 72].

#### *4.2.3. Wnt pathways*

Activation of Wnt canonical pathways involves the formation of complexes between Wnt1, or Wnt3a, Frizzled proteins and LRP-5/6, which may be integrin dependent. Those complexes prevent cytoplasmic β-catenin degradation, which, in turn, translocates to nucleus, where it activates members of the TCF/LEF family to promote osteogenesis. Acoustic stimulation increases expression of Wnt1, Wnt3a, β-catenin and Frizzled proteins 2/4. It also activates βcatenin in an integrin-dependent manner. Wnt5a, which plays a role in Wnt non-canonical pathway, is responsive to mechanical stimulation, but its responsiveness to acoustic stimula‐ tion is yet to be evaluated [3, 10, 73].

#### *4.2.4. Growth factors and hormones crosstalk*

Different growth factors and hormones induce osteogenesis that includes bone cells prolifer‐ ation, migration and adhesion to stimulation sites, angiogenesis and osteogenic differentiation. Mechanical stimulation possesses the same effect and affects growth factors and hormones signalling. Acoustic stimulation increases the expression of BMP-2/4/7 and related receptors (BMPR-IA, BMPR-IB, ActR-I, BMPR-II, ActR-IIA, ActR-IIB), FGF-2, IGF-1, PTHr-1, TGF-β1 and VEGF. Nevertheless, the exact signaling pathways of those factors are still not fully understood [37, 44, 54, 66, 74–80].

BMP-2, BMP-4 and BMP-7 play important roles in osteogenesis following fracture. They stimulate mesenchymal cell proliferation and osteogenic differentiation, induce osteoproge‐ nitor cell migration, modulate osteoclast activity and promote angiogenesis. Their mechanism of action involves Smad-1, which is activated by BMP receptors; then Smad-1 translocates to nucleus where it upregulates transcription of osteogenic factors as cbfa1. Acoustic stimulation activates Smad-1, but it has not been proved whether BMP receptors activity is responsible to Smad-1 acoustically induced activation [66, 75].

Similar to BMPs, TGF-β1 induces cellular proliferation, osteogenic differentiation, minerali‐ zation and angiogenesis. Acoustic force-induced TGF-β1 production depends on superoxide production which is possibly promoted by Ras/Rac-1/NADPH oxidase pathway. Superoxide is a free radical that, in contrary to common knowledge, is harmless to bone cells when produced by a certain range of acoustic pressure (that is yet to be determined). Moreover, superoxide promotes ERK activation, which induces osteogenic differentiation through cbfa-1 transcription [79, 81].

Angiogenesis is vital for fracture healing. BMPs, TGF-β1 and VEGF induce angiogenesis. Among those factors, VEGF seems to be the most important for angiogenesis. HIF-1α is a transcription factor that regulates VEGF expression and is activated by acoustic stimulation. In addition, superoxide and Ras mediate HIF-1α activation and VEGF expression. However, VEGF expression is not dependent on BMP-2, TGF-β1, IGF-1, cox-2, PGE2 and Ca2+ influx. Interestingly, LIPUS-induced VEGF expression depends on NO production, whereas ESWTinduced VEGF expression does not [81–83].

#### *4.2.5. Differentiation markers and transcriptional factors*

A variety of differentiation markers are modulated by acoustic stimulation, such as cbfa-1, osterix, bone sialoprotein, osteopontin, osteocalcin, type I collagen and ALP. Contrarily, the unique report investigating RPWT effects in osteoblasts showed decreased expression of cbfa-1, osterix, type I collagen, bone sialoprotein and osteocalcin. RPWT is commonly used for orthopaedic pathologies of soft tissues with satisfactory results, but no reports were found for bone-related orthopaedic disorders. Therefore, further investigations are required to deter‐ mine the biological effects of RPWT on bone [33, 43, 44, 75, 76].

Regarding cellular proliferation, there are some transcriptional factors that are affected by acoustic forces, such as c-fos, c-jun, c-myc, egr-1, TSC-22, SOST, FGF-23, Dlx, Msx2 and cyclin E2/CDK2 [37, 39, 43, 55, 68, 69, 84–86].

#### *4.2.6. Signalling for migration*

*4.2.2. ATP receptors*

40 Advanced Techniques in Bone Regeneration

[3, 64, 72].

*4.2.3. Wnt pathways*

[37, 44, 54, 66, 74–80].

transcription [79, 81].

tion is yet to be evaluated [3, 10, 73].

*4.2.4. Growth factors and hormones crosstalk*

Smad-1 acoustically induced activation [66, 75].

ATP receptors promote the exchange of calcium from intracellular deposits to extracellular environment, or from extracellular environment to intracellular environment. ATP receptors complex with integrins and G proteins, and some (P2X7 and P2Y1) are activated by mechanical loading. By means that need to be explored, mechanically induced activation of P2X7 and P2Y1 induce osteogenic differentiation—represented by increased expression of cbfa-1, osterix, type I collagen, bone sialoprotein, osteopontin and osteocalcin—and osteoblasts proliferation

Activation of Wnt canonical pathways involves the formation of complexes between Wnt1, or Wnt3a, Frizzled proteins and LRP-5/6, which may be integrin dependent. Those complexes prevent cytoplasmic β-catenin degradation, which, in turn, translocates to nucleus, where it activates members of the TCF/LEF family to promote osteogenesis. Acoustic stimulation increases expression of Wnt1, Wnt3a, β-catenin and Frizzled proteins 2/4. It also activates βcatenin in an integrin-dependent manner. Wnt5a, which plays a role in Wnt non-canonical pathway, is responsive to mechanical stimulation, but its responsiveness to acoustic stimula‐

Different growth factors and hormones induce osteogenesis that includes bone cells prolifer‐ ation, migration and adhesion to stimulation sites, angiogenesis and osteogenic differentiation. Mechanical stimulation possesses the same effect and affects growth factors and hormones signalling. Acoustic stimulation increases the expression of BMP-2/4/7 and related receptors (BMPR-IA, BMPR-IB, ActR-I, BMPR-II, ActR-IIA, ActR-IIB), FGF-2, IGF-1, PTHr-1, TGF-β1 and VEGF. Nevertheless, the exact signaling pathways of those factors are still not fully understood

BMP-2, BMP-4 and BMP-7 play important roles in osteogenesis following fracture. They stimulate mesenchymal cell proliferation and osteogenic differentiation, induce osteoproge‐ nitor cell migration, modulate osteoclast activity and promote angiogenesis. Their mechanism of action involves Smad-1, which is activated by BMP receptors; then Smad-1 translocates to nucleus where it upregulates transcription of osteogenic factors as cbfa1. Acoustic stimulation activates Smad-1, but it has not been proved whether BMP receptors activity is responsible to

Similar to BMPs, TGF-β1 induces cellular proliferation, osteogenic differentiation, minerali‐ zation and angiogenesis. Acoustic force-induced TGF-β1 production depends on superoxide production which is possibly promoted by Ras/Rac-1/NADPH oxidase pathway. Superoxide is a free radical that, in contrary to common knowledge, is harmless to bone cells when produced by a certain range of acoustic pressure (that is yet to be determined). Moreover, superoxide promotes ERK activation, which induces osteogenic differentiation through cbfa-1

Cells must migrate to the healing site so that new bone can be generated. SDF-1 is an important chemotactic factor mostly produced by immature osteoblasts in the endosteal region near stem cells population. SDF-1 normally is released from the fracture site to attract mesenchymal stem cells which will differentiate into osteoblasts. SDF-1 binds to CXCR4, a seven transmembrane G-protein coupled receptor, and triggers a cascade of reactions leading to cellular migration and survival. Reports have shown that acoustic loading increases expression of SDF-1 and CXCR4, thereby resulting in mesenchymal stem cells migration to the fracture site. In spite of that, more investigation is needed to clarify the exact cascade of reactions triggered by SFD-1/ CXCR4 [56, 87–89].

#### *4.2.7. Signalling for bone remodelling*

Bone remodelling is an important step of bone healing. This important stage follows bone formation and is governed by osteoclasts—bone cells of the granulocyte/monocyte lineage that resorb extracellular matrix. In order to attract osteoclast progenitor cells to the healing site, osteoblasts express MCP-1, MIP-1, RANTES and IL-8. Osteoblasts also express, or secrete, RANKL, which induces osteoclasts differentiation through their native RANK; and secrete OPG, a decoy receptor of RANKL, which antagonizes RANKL-mediated osteoclastogenesis. Acoustic loading in the form of LIPUS affects osteoclastogenesis by increasing the expression of MCP-1, MIP-1b, RANKL and OPG in osteoblasts through AT1. Increased RANKL expres‐ sion is also dependent on integrins activity. Moreover, acoustic forces increase the expression of MIP-2, which may also be involved in osteoclastogenesis. On the other hand, it has been shown that low-energy ESWT decreases OPG and RANKL expression in osteoblasts; RPWT does not change OPG expression, but decreases RANKL; and LIPUS does not change the expression of OPG (contradictory results) and RANTES in osteoblasts. Those data show that acoustic deformation affects osteoclastogenesis; however, the exact influence on osteoclasto‐ genesis needs to be better elucidated [33, 43, 50, 55].

#### *4.2.8. Proteins with few data*

There is another list of proteins whose activation has been shown to be influenced by acoustic waves, but there is poor information about their role in mechanotransduction and osteogen‐ esis:

