**5. Optimization of biological responses**

As previously described, acoustic loads can be exerted by different types of waves, such as LIPUS, ESWT and RPWT. Changing some physical properties (e.g. magnitude and frequency) and mode of application (e.g. axial distance, incidence angle and number of cycles) of acoustic waves can elicit different cellular responses. No studies explored the subject with RPWT.

#### **5.1. Magnitude and number of cycles**

According to mechanostat model, for strains within the overuse range, bone formation increases as a proportion of the load magnitude. Loads within the pathological overuse range stimulate osteogenesis, but also damage tissue until bone breaks (about 15,000–20,000 μstrain). Furthermore, cellular response also increases as a proportion of the number of cycles [3].

LIPUS at intensities between 2 and 150 mW/cm2 were compared. Higher intensities produced greater bone formation, faster healing rate, and better torsional stiffness and failure torque. The best results were found for 30 mW/cm2 . Average temperature at the soft tissue was 1.74°C higher for 150 mW/cm2 in comparison with 30 mW/cm2 . Temperature elevation may affect some enzymes like collagenase I and cause tissue damage, resulting in worse biological response. LIPUS commonly is applied as a daily 20-min treatment; therefore, the number of cycles are not changed [29, 93, 94].

On the contrary, there is no exact protocol for ESWT that determines the best response to stimulation. ESWT at magnitudes ranging from 0.05 to 0.62 mJ/mm2 positively affects osteo‐ genesis. The number of impulses of shock waves corresponds to the number of cycles of ESWT. In studies, number of impulses varies from 250 to more than 4000. Moreover, biological response is different when treatment is performed in vitro, or in vivo with small animals (e.g. rodents), or in vivo with large animals (e.g. goats) and humans. For most in vitro studies, 500 impulses promote the best cellular response; above this threshold, cellular damage surpasses bone formation. On the other hand, the best intensity (mJ/mm2 ) could not be found, suggesting that, for cells directly exposed to ESWT, the number of cycles affects cellular response more than the intensity of energy density itself. On the other hand, most in vivo studies in animals show that better responses are elicited by higher energy densities up to 0.47 mJ/mm2 in comparative studies, while number of cycles (impulses) was not proved to have the same influence [54, 95–98].

#### **5.2. Frequency**

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‐

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‐

**1.** AT1 is classically involved in arterial pressure control. This receptor was identified in bone cells, but its role is yet to be determined. AT1 is required for mechanically induced ERK-1/2

**2.** Bax is a key component for apoptosis induced through interactions with pore proteins on the mitochondrial membrane. Bax mechanism of activation is complex and not fully understood, but may be modulated by acoustic deformation in favour of cell survival. Bax

**3.** IRS-1 activity increased in intact and healthy bones of rats subjected to acoustic stimula‐ tion. IRS-1 is involved in insulin-mediated and IGF-1-mediated bone formation, but its

**4.** p38 is a MAPK that regulates cell proliferation and differentiation. Because conflicting results were found for p38 activation following LIPUS and ESWT, more investigation is required. Some studies report increased activity, while others report unchanged activity

**5.** PPARγ2 is expressed in mesenchymal stem cells. Upon acoustic stimulation, PPARγ2

As previously described, acoustic loads can be exerted by different types of waves, such as LIPUS, ESWT and RPWT. Changing some physical properties (e.g. magnitude and frequency) and mode of application (e.g. axial distance, incidence angle and number of cycles) of acoustic waves can elicit different cellular responses. No studies explored the subject with RPWT.

According to mechanostat model, for strains within the overuse range, bone formation increases as a proportion of the load magnitude. Loads within the pathological overuse range

drives those cells to differentiate into osteogenic lineage [68].

mechanism of activation following acoustic loading is yet to be determined [90].

genesis needs to be better elucidated [33, 43, 50, 55].

activation is also integrin dependent [43, 70].

**5. Optimization of biological responses**

**5.1. Magnitude and number of cycles**

*4.2.8. Proteins with few data*

42 Advanced Techniques in Bone Regeneration

activation [50].

[68, 81, 91, 92].

esis:

Normally, load frequencies within the range of 1–30 Hz at physiological and overuse ranges progressively induce osteogenesis. Higher frequencies (17–90 Hz), in the form of vibration, induce osteogenesis but at a much lower strain range (about 5 μstrain; i.e. strain in the order of 10−5). LIPUS is a low magnitude and high frequency wave, which, based on mathematical and experimental models, produces strains in the order of 10−5 at 1.5 MHz. Because of high frequency, those strains promote the same effects as strains in the order of 10−1 (i.e. 10% = 100,000 μstrain) at 1 Hz on cells, and the estimated intracellular strain on organelles is about 0.5% (i.e. 5000 μstrain). Accordingly, it was shown that LIPUS increased transcriptional factors (c-fos, c-jun and c-myc) as frequency increased, resulting in maximum response at 5 MHz (within a range from 2 to 8 Mhz). Those calculations were obtained for strains at cellular level. For strains at bone level, it is believed that the model for strain amplification (see Section 4.1) may apply firstly, followed by the estimative presented here. No investigations were found about the role of frequency on ESWT and RPWT [3, 99–101].

#### **5.3. Axial distance**

Energy distribution varies according to the distance from the transducer and the surface (axial distance). For LIPUS, two zones were defined according to axial distance: near field (close to transducer) and far field (about 130 mm away from transducer). There is also a mid-near field, when the surface is about 60 mm away from transducer. Within near field, energy distribution of LIPUS beam is not uniform. As such, there are many peaks of acoustic pressure (maxima and minima) across the beam diameter. As the distance from transducer increases, the number of peaks of acoustic pressure across the beam diameter decreases (less maxima and minima). When surface is at far field, a regular beam is formed [5, 102].

As LIPUS transducer is placed transcutaneously during treatment, superficial and deeper cells are exposed to different acoustic fields. Although LIPUS promotes osteogenesis within near, mid-near and far fields, axial distance affects the biological effects of LIPUS. Mid-near field LIPUS elicited greater callus formation in a fractured-femur rat model; on the other hand, in that same model, femurs subjected to far field LIPUS exhibited higher peak torque and torsional stiffness. Those results indicate that, mid-near field LIPUS is optimal for cellular proliferation, while far field LIPUS stands for osteogenic differentiation (bone mineralization). Reinforcing that, mid-near LIPUS incited more NO production whereas far field LIPUS promoted increased ALP activity and mineralization in preosteoblasts. Moreover, both midnear and far field LIPUS produced increased β-catenin nuclear translocation [5, 102].

During ESWT, maximal intensity of energy density is obtained at the focus. Consequently, superficial and deeper cells are exposed to different acoustic fields. However, no studies were found on this subject for ESWT and RPWT.

#### **5.4. Incidence angle**

As previously described, acoustic waves transmitted into bone can be decomposed in longi‐ tudinal waves and shear waves. The magnitude of each wave depends on the incidence angle of the acoustic wave. Accordingly, two critical angles were determined. The first critical angle is defined as the angle of incidence after which incident acoustic waves travel along the medium surface and only shear waves are refracted to that medium. In that case, longitudinal waves do not travel into the medium. The second critical angle is defined as the angle at which acoustic waves are totally reflected and shear waves travel along the medium surface, but not into the medium. For LIPUS, the first critical angle is 22°, and the second critical angle is 48°. Between the first and second critical angles, at 35°, the amount of transmitted shear waves is maximized, and an optimal cellular response is obtained. Those critical angles were not determined for ESWT and RPWT [36].

#### **5.5. Different sources of acoustic waves**

As previously described, the method for producing low-intensity pulsed ultrasound waves is unique, but there are three generation methods for extracorporeal shock waves. No clinical studies compare the effectiveness between the three methods, but one experimental research compared osteoblasts responses to electrohydraulic and electromagnetic ESWT. It was found greater cell viability and osteocalcin expression for electrohydraulic-stimulated cells, and greater expression of type I procollagen-C enzyme, and TGF-β1 production for electromag‐ netic-stimulated cells. These findings can be attributed to the difference in the pressure distribution at the focal zone between the electrohydraulic and electromagnetic generators [40].

#### **5.6. Combined therapy**

transducer) and far field (about 130 mm away from transducer). There is also a mid-near field, when the surface is about 60 mm away from transducer. Within near field, energy distribution of LIPUS beam is not uniform. As such, there are many peaks of acoustic pressure (maxima and minima) across the beam diameter. As the distance from transducer increases, the number of peaks of acoustic pressure across the beam diameter decreases (less maxima and minima).

As LIPUS transducer is placed transcutaneously during treatment, superficial and deeper cells are exposed to different acoustic fields. Although LIPUS promotes osteogenesis within near, mid-near and far fields, axial distance affects the biological effects of LIPUS. Mid-near field LIPUS elicited greater callus formation in a fractured-femur rat model; on the other hand, in that same model, femurs subjected to far field LIPUS exhibited higher peak torque and torsional stiffness. Those results indicate that, mid-near field LIPUS is optimal for cellular proliferation, while far field LIPUS stands for osteogenic differentiation (bone mineralization). Reinforcing that, mid-near LIPUS incited more NO production whereas far field LIPUS promoted increased ALP activity and mineralization in preosteoblasts. Moreover, both mid-

near and far field LIPUS produced increased β-catenin nuclear translocation [5, 102].

During ESWT, maximal intensity of energy density is obtained at the focus. Consequently, superficial and deeper cells are exposed to different acoustic fields. However, no studies were

As previously described, acoustic waves transmitted into bone can be decomposed in longi‐ tudinal waves and shear waves. The magnitude of each wave depends on the incidence angle of the acoustic wave. Accordingly, two critical angles were determined. The first critical angle is defined as the angle of incidence after which incident acoustic waves travel along the medium surface and only shear waves are refracted to that medium. In that case, longitudinal waves do not travel into the medium. The second critical angle is defined as the angle at which acoustic waves are totally reflected and shear waves travel along the medium surface, but not into the medium. For LIPUS, the first critical angle is 22°, and the second critical angle is 48°. Between the first and second critical angles, at 35°, the amount of transmitted shear waves is maximized, and an optimal cellular response is obtained. Those critical angles were not

As previously described, the method for producing low-intensity pulsed ultrasound waves is unique, but there are three generation methods for extracorporeal shock waves. No clinical studies compare the effectiveness between the three methods, but one experimental research compared osteoblasts responses to electrohydraulic and electromagnetic ESWT. It was found greater cell viability and osteocalcin expression for electrohydraulic-stimulated cells, and greater expression of type I procollagen-C enzyme, and TGF-β1 production for electromag‐ netic-stimulated cells. These findings can be attributed to the difference in the pressure

When surface is at far field, a regular beam is formed [5, 102].

found on this subject for ESWT and RPWT.

44 Advanced Techniques in Bone Regeneration

determined for ESWT and RPWT [36].

**5.5. Different sources of acoustic waves**

**5.4. Incidence angle**

Mechanical stimulation can be combined with different types of acoustic waves or with growth factors.

### *5.6.1. ESWT and LIPUS*

Electromagnetic ESWT and LIPUS combined therapy applied to periosteal cells showed no difference regarding cell proliferation, cell viability and ALP activity in comparison with ESWT alone, but, in comparison with LIPUS alone, showed worse results for early response (after 6 days) and better results for late response (after 18 days) [42].

#### *5.6.2. LIPUS and growth factors*

BMPs are known osteogenic factors. Their combined therapy (BMP-7 or rhBMP-2) with LIPUS enhances bone formation, osteogenic differentiation and biomechanical properties of bone [103, 104].

Bisphosphonates are anti-osteoclastic agents that increase or maintain bone mineral density in osteoporotic patients. Combined therapy with LIPUS is not better than alendronate or LIPUS alone to increase bone healing. On the other hand, combined therapy with LIPUS enhances bone mineral density more than separate treatment [105].

1,25-Dihydroxyvitamin D3 increases the expression of VEGF in osteoblasts and modulates cellular proliferation and differentiation. Combined treatment with LIPUS, however, does not ameliorate cellular response in comparison with LIPUS or 1,25-dihydroxyvitamin D3 alone [106].

Statins (e.g. simvastatin, mevastatin and lovastatin) stimulate osteogenesis through Ras/Smad/ ERK/BMP-2 pathway. Combined therapy with LIPUS does not increase bone formation rate more than statins or LIPUS alone [77].

No studies were found on the subject for ESWT and RPWT.
