1 3 Stimulation of bone repair with ultrasound: A review of the possible 4 mechanic effects 5 6 7 Frédéric Padilla a,b,⇑,1 Q1 , Regina Puts c,⇑,1 , Laurence Vico d , Kay Raum c 8 a Inserm, U1032, LabTau, Lyon F-69003, France 9 b Université de Lyon, Lyon F-69003, France 10 c Julius Wolff Institut & Berlin-Brandenburg School for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Germany 11 d Inserm U1059 Lab Biologie intégrée du Tissu Osseux, Université de Lyon, St-Etienne F-42023, France 12 13 15 article info 16 Article history: 17 Available online xxxx 18 Keywords: 19 Aco Q4 ustic streaming 20 Bioeffects 21 Bone 22 BMP 23 Differentiation 24 Fracture 25 Growth factors 26 Healing 27 LIPUS 28 MSC 29 Mechansensation 30 Mechanotransduction 31 Osteoblasts 32 Proliferation 33 Radiation force 34 Stem cells 35 Stimulation 36 Shock waves 37 Ultrasound 38 39 abstract 40 In vivo and in vitro studies have demonstrated the positive role that ultrasound can play in the enhance- 41 ment of fracture healing or in the reactivation of a failed healing process. We review the several options 42 available for the use of ultrasound in this context, either to induce a direct physical effect (LIPUS, shock 43 waves), to deliver bioactive molecules such as growth factors, or to transfect cells with osteogenic plas- 44 mids; with a main focus on LIPUS (or Low Intensity Pulsed Ultrasound) as it is the most widespread and 45 studied technique. The biological response to LIPUS is complex as numerous cell types respond to this 46 stimulus involving several pathways. Known to-date mechanotransduction pathways involved in cell 47 responses include MAPK and other kinases signaling pathways, gap-junctional intercellular communica- 48 tion, up-regulation and clustering of integrins, involvement of the COX-2/PGE2, iNOS/NO pathways and 49 activation of ATI mechanoreceptor. The mechanisms by which ultrasound can trigger these effects remain 50 intriguing. Possible mechanisms include direct and indirect mechanical effects like acoustic radiation 51 force, acoustic streaming, and propagation of surface waves, fluid-flow induced circulation and redistri- 52 bution of nutrients, oxygen and signaling molecules. Effects caused by the transformation of acoustic 53 wave energy into heat can usually be neglected, but heating of the transducer may have a potential 54 impact on the stimulation in some in-vitro systems, depending on the coupling conditions. Cavitation 55 cannot occur at the pressure levels delivered by LIPUS. In-vitro studies, although not appropriate to iden- 56 tify the overall biological effects, are of great interest to study specific mechanisms of action. The diver- 57 sity of current experimental set-ups however renders this analysis very complex, as phenomena such as 58 transducer heating, inhomogeneities of the sound intensity in the near field, resonances in the transmis- 59 sion and reflection through the culture dish walls and the formation of standing waves will greatly affect 60 the local type and amplitude of the stimulus exerted on the cells. A future engineering challenge is there- 61 fore the design of dedicated experimental set-ups, in which the different mechanical phenomena induced 62 by ultrasound can be controlled. This is a prerequisite to evaluate the biological effects of the different 63 phenomena with respect to particular parameters, like intensity, frequency, or duty cycle. By relating 64 the variations of these parameters to the induced physical effects and to the biological responses, it will 65 become possible to derive an ‘acoustic dose’ and propose a quantification and cross-calibration of the dif- 66 ferent experimental systems. Improvements in bone healing management will probably also come from a 67 combination of ultrasound with a ‘biologic’ components, e.g. growth factors, scaffolds, gene therapies, or 68 drug delivery vehicles, the effects of which being potentiated by the ultrasound. 69 Ó 2014 Published by Elsevier B.V. 70 71 72 1. Introduction 73 There are several ways by which ultrasound can influence bone 74 fracture healing. Ultrasound has played, or has the potential to 75 play, a role in different aspects of the process of bone regeneration. 76 It can act on the biologics components of the regeneration process 77 via promotion of cell proliferation, cells pre-conditioning to orient 78 their differentiation during culture [1,2], or cells transfection [3]. 79 Ultrasound can modulate the micro-environment by triggering 80 delivery of growth factors or gene expression in engineered cells 81 [4,5]; or by modulating the physical environment by heat deposi- 82 tion or mechanical stimulation [6]. Ultrasound can also be useful 83 in tissue engineering approaches by acting on the scaffolds for 84 improvements of scaffold integration, characterization and control 0041-624X/$ - see front matter Ó 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ultras.2014.01.004 ⇑ Correspo Q2 nding author. Tel.: +33 472681918. E-mail address: [email protected](F. Padilla). 1 Equal contributors. Ultrasonics xxx (2014) xxx–xxx Contents lists available at ScienceDirect Ultrasonics journal homepage: www.elsevier.com/locate/ultras ULTRAS 4744 No. of Pages 21, Model 5G 21 January 2014 Please cite this article in press as: F. Padilla et al., Stimulation of bone repair with ultrasound: A review of the possible mechanic effects, Ultrasonics (2014), http://dx.doi.org/10.1016/j.ultras.2014.01.004
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Contents lists available at ScienceDirect
Ultrasonics
journal homepage: www.elsevier .com/locate /ul t ras
Stimulation of bone repair with ultrasound: A review of the possiblemechanic effects
0041-624X/$ - see front matter � 2014 Published by Elsevier B.V.http://dx.doi.org/10.1016/j.ultras.2014.01.004
Please cite this article in press as: F. Padilla et al., Stimulation of bone repair with ultrasound: A review of the possible mechanic effects, Ultrasonicshttp://dx.doi.org/10.1016/j.ultras.2014.01.004
Frédéric Padilla a,b,⇑,1, Regina Puts c,⇑,1, Laurence Vico d, Kay Raum c
a Inserm, U1032, LabTau, Lyon F-69003, Franceb Université de Lyon, Lyon F-69003, Francec Julius Wolff Institut & Berlin-Brandenburg School for Regenerative Therapies, Charité - Universitätsmedizin Berlin, Germanyd Inserm U1059 Lab Biologie intégrée du Tissu Osseux, Université de Lyon, St-Etienne F-42023, France
In vivo and in vitro studies have demonstrated the positive role that ultrasound can play in the enhance-ment of fracture healing or in the reactivation of a failed healing process. We review the several optionsavailable for the use of ultrasound in this context, either to induce a direct physical effect (LIPUS, shockwaves), to deliver bioactive molecules such as growth factors, or to transfect cells with osteogenic plas-mids; with a main focus on LIPUS (or Low Intensity Pulsed Ultrasound) as it is the most widespread andstudied technique. The biological response to LIPUS is complex as numerous cell types respond to thisstimulus involving several pathways. Known to-date mechanotransduction pathways involved in cellresponses include MAPK and other kinases signaling pathways, gap-junctional intercellular communica-tion, up-regulation and clustering of integrins, involvement of the COX-2/PGE2, iNOS/NO pathways andactivation of ATI mechanoreceptor. The mechanisms by which ultrasound can trigger these effects remainintriguing. Possible mechanisms include direct and indirect mechanical effects like acoustic radiationforce, acoustic streaming, and propagation of surface waves, fluid-flow induced circulation and redistri-bution of nutrients, oxygen and signaling molecules. Effects caused by the transformation of acousticwave energy into heat can usually be neglected, but heating of the transducer may have a potentialimpact on the stimulation in some in-vitro systems, depending on the coupling conditions. Cavitationcannot occur at the pressure levels delivered by LIPUS. In-vitro studies, although not appropriate to iden-tify the overall biological effects, are of great interest to study specific mechanisms of action. The diver-sity of current experimental set-ups however renders this analysis very complex, as phenomena such astransducer heating, inhomogeneities of the sound intensity in the near field, resonances in the transmis-sion and reflection through the culture dish walls and the formation of standing waves will greatly affectthe local type and amplitude of the stimulus exerted on the cells. A future engineering challenge is there-fore the design of dedicated experimental set-ups, in which the different mechanical phenomena inducedby ultrasound can be controlled. This is a prerequisite to evaluate the biological effects of the differentphenomena with respect to particular parameters, like intensity, frequency, or duty cycle. By relatingthe variations of these parameters to the induced physical effects and to the biological responses, it willbecome possible to derive an ‘acoustic dose’ and propose a quantification and cross-calibration of the dif-ferent experimental systems. Improvements in bone healing management will probably also come from acombination of ultrasound with a ‘biologic’ components, e.g. growth factors, scaffolds, gene therapies, ordrug delivery vehicles, the effects of which being potentiated by the ultrasound.
� 2014 Published by Elsevier B.V.
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1. Introduction
There are several ways by which ultrasound can influence bonefracture healing. Ultrasound has played, or has the potential toplay, a role in different aspects of the process of bone regeneration.
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It can act on the biologics components of the regeneration processvia promotion of cell proliferation, cells pre-conditioning to orienttheir differentiation during culture [1,2], or cells transfection [3].Ultrasound can modulate the micro-environment by triggeringdelivery of growth factors or gene expression in engineered cells[4,5]; or by modulating the physical environment by heat deposi-tion or mechanical stimulation [6]. Ultrasound can also be usefulin tissue engineering approaches by acting on the scaffolds forimprovements of scaffold integration, characterization and control
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of the rate of scaffold degradation [7–10]. Within this arsenal, theLIPUS (or Low Intensity Pulsed Ultrasound) techniques aim at mod-ulating the physical environment of the cells, in particular bymechanical stimulation.
Different forms of ultrasound treatment (LIPUS, Shock Waves)have been proposed to stimulate or induce bone repair. Biophysicaleffects of ultrasound, and in particular of therapeutic ultrasoundused for thermal ablation or drug delivery, have been fairly docu-mented [11]. However, the mechanisms by which ultrasound caninteract with cells and/or their microenvironments during fracturehealing are still open to debate.
Clinical results obtained with ultrasound stimulation of bonehealing are still controversial, suggesting a potential effective rolebut depending of the medical history of previous treatments, siteand type of fracture or bone loss (like bone lengthening), pathology(fresh fracture vs. delayed unions) and treatment modality (treat-ment daily duration, intensity, frequency, etc.), suggesting the needfor standardization of treatment dose and for further randomizedcontrolled trials [12,1,13,14]. Moreover, the lack of understandingof the relevant mechanisms that triggers a positive biological re-sponse suggests that optimization of devices’ technology and treat-ment regimen remains to be fulfilled.
The main purpose of this review is to give the reader a generalidea on existing ultrasound applications for the stimulation of bonehealing and treatment of non-unions. The main focus is placed onthe LIPUS technique, which has pronounced bioeffects on tissuesregeneration, while employing intensities within a diagnosticrange [1,13,15]. The updated state of the LIPUS biological knowl-edge is summarized and discussed through the prism of plausiblephysical effects implicated with observed biological phenomena.
2. Basics of Biomedical Ultrasound
The term ultrasound refers to the propagation of an acousticwave, i.e. a travelling mechanical perturbation, whose frequencyis above the audible range, typically from a few tenth of kHz to sev-eral tenths of MHz. Ultrasound in liquid and in soft tissues usuallyrefers to the propagation of a longitudinal wave, causing locallyos-cillatory motions of particles around their initial positions. Thiswill result in local changes of the medium’s density and pressure,an increase in location of rarefraction (low pressure) and increasein the location of compression (high pressure) cycles of the wave.Depending on the frequency, level of acoustical energy and /orpressure emitted by the source, applications can be categorizedas diagnostic or therapeutic, where diagnostic intensities (spatiallyand temporarily averaged) are typically below 100 mW/cm2.
Diagnostic applications include gray-scale imaging or ‘B-mode’imaging or echography, where the variations in the amplitude ofthe backscattered ultrasonic waves by tissues are displayed as afunction of depth to visualize morphologic features or to analyzestructure of the tissues [16]; and elastography for direct imagingof the strain and Young’s or shear modulus of tissues, based on aultrasonic measure of internal tissue motions as a results of theapplication of a mechanical stimulus [17]. Even if diagnostic ultra-sound can potentially induce some biological effects [18], in partic-ular in the presence of ultrasound contrast agents [19], indices likethe mechanical and thermal indexes are used to stay within arange of energy/intensity deposition that will avoid them. Theseimaging technologies can have applications in the characterizationof the bone healing process [20,21]. In bone tissue engineering,elastography has also been proposed as a way to monitor scaffolddegradation [22].
In contrary to diagnostic applications, therapeutic ultrasound ispurposely looking for the induction of bio-effects in tissues. Thereare several ways by which ultrasound can interact with tissues to
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induce biological effects [23,24], relying on thermal effects for heatdeposition, or non-thermal effects like cavitation or radiation force.
Ultrasound energy absorption is used to elevate temperature intissues, and high intensity focused ultrasound or HIFU is used tothermally ablate cancer tumors [25,26]. In a more moderate re-gime, ultrasound energy delivery can also be used to induce mildhyperthermia and is used in physiotherapy to promote healing ofboth bone and soft tissues [27]. Heat deposition can also be usedto control the expression of reporter genes under transcriptionalcontrol of a heat-inducible promoter [28]. In shock wave litho-tripsy, shock waves, generated outside the body, are focused to afixed location to produce locally very large acoustic pressures,inducing kidney stones comminution [29]. Ultrasound can also in-duce temporary cell membrane permeability, a phenomenoncalled sonoporation [30], and this approach can be used to enhancedrug or genetic material uptake [31]. For these effects, the principlemechanism is believed to be cavitation, the growth, oscillation, andeventually collapse of gas bubbles in liquid driven by an ultrasoundwave. Combined with thermo- or mechanical-sensitive carriers,like thermo-sensitive liposomes [32], echogenic liposomes [33]or superheated perfluorocarbone droplets [34], ultrasound can beused to control spatially the release of drugs by inducing ruptureor pore-like defects in carriers membranes. DNA delivery into var-ious cells in vitro and in vivo has also been reported [35], demon-strating increased gene expression, even if optimization is stillrequired to achieve high transfection rates [36]. Other therapeuticapplications of ultrasound have been developed, such as sono-thrombolysis, or ultrasound cutting but are so far not used in appli-cations related to tissue engineering. The reader will find someinsight in the review papers [23,37–39].
In between the diagnostic and therapeutic regimes lies the so-called low-intensity pulsed ultrasound technique or LIPUS, wherelow ultrasound intensity, typically below 100 mW/cm2, i.e. at theupper limit of diagnostic intensities, is delivered in a pulsed man-ner. In contrast to a diagnostic application, the ultrasound is deliv-ered in a pulsed manner with long duty cycles and exposure times.LIPUS have been reported to be responsible for several biologicalreactions in vitro and in vivo, in particular enhancing the healingrate of bone fractures [1] and soft tissue healing [40], with reportedcellular responses that we will analyze later in this review.
3. LIPUS physics
3.1. LIPUS exposure conditions
Heating, cavitation and acoustic streaming have been proposedto be the main physical mechanisms to stimulate cells in vitro. LI-PUS stimulation studies have been conducted with frequencies be-tween 45 kHz and 3 MHz, intensity levels between 5 and 1000 mW/cm2 (SATA: spatial average, time average), in continuous or burstmode, and with daily exposure times between 1 and 20 min.
The vast majority of the published studies were performed withdevices similar to the commercial system Exogen (SAFHS, Exogen,NJ). This system uses unfocused circular transducers with effectivesurface areas of 3.88 cm2 and the following typical stimulationconditions: frequency 1.5 MHz, intensity 30 mW/cm2 (SATA), burstmode 200 ls ON/800 ls OFF (i.e. pulse repetition rate 1 kHz), dailyexposure: 20 min [41]. Other studies report the use of unfocusedtransducers with different surface areas, ultrasound frequenciesbetween 45 kHz [42] and 3 MHz [43], intensity levels between 5and 2400 mW/cm2 [42,44], and duty cycles (e.g. 2 ms ON at100 Hz pulse repetition rate, or continuous wave mode) [43].
For lossless linear plane wave propagation the relation betweenacoustic impedance Z = qc, particle velocity m and acoustic pressureP is:
h ultrasound: A review of the possible mechanic effects, Ultrasonics (2014),
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P ¼ Z � m: ð1Þ
The intensity is proportional to the square of the acousticpressure:
I ¼ jpr j2
2Z; ð2Þ
where |pr|2 is the absolute acoustic peak rarefractional pressure ofthe wave. The mechanical index MI is defined as:
MI ¼ prffiffiffiffif0
p ; ð3Þ
whereas f0 is the frequency of the acoustic wave. The spatial inten-sity distribution of a plane circular transducer is shown in Fig. 1. ForLIPUS studies, the spatially and temporally averaged intensity(ISATA) is commonly reported. The latter is determined by meansof the acoustic radiation force Frad acting on a large absorber thatis placed perpendicular to the beam axis in the sound field (i.e. aradiation force balance):
Frad ¼Wc0; ð4Þ
whereas W and c0 are the total acoustic output power and thesound velocity in the medium, respectively. The force can be mea-sured with an ultrasonic power balance. For example, the acousticoutput power of 1 mW produces a force of 0.69 lN in water [45].Total acoustic output power is related to the transducer surface areaa, ISATA by:
ISATA ¼W=a: ð5Þ
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3.2. Rationale for LIPUS
It is important to consider the spatial and temporal characteris-tics of the acoustic waveform used in LIPUS studies. For a continu-ous wave the radiation force is time invariant. However, for pulsedexcitation the force varies periodically at the pulse repetition fre-quency (i.e. the inverse of the period between the emissions oftwo consecutive pulses). For the commonly used burst excitationat 1 kHz pulse repetition frequency (son = 200 ls), the relationbetween temporal peak (ITP) and temporal averaged (ITA)intensity is:
ITA ¼ son � PRF � ITP ¼ 0:2 � ITP: ð6Þ
The spatial and temporal variations of ITA within the acousticbeam give rise to local radiation pressure variations and resultingstrain and motion at frequencies independent of the emissionacoustic frequency but related to the PRF. The local radiation pres-sure averaged over the pulse duration son is:
Prad ¼12jprj
2
q0c20
¼ IPA
c0: ð7Þ
Fig. 1. (a) Temporal peak intensity (ITP) distribution of a plane circular transducer of 22 mthe x, y plane. (b) Axial intensity (ITP) distribution along the beam axis (x, y = 0). (c) Late
Please cite this article in press as: F. Padilla et al., Stimulation of bone repair withttp://dx.doi.org/10.1016/j.ultras.2014.01.004
IPA denotes intensity averaged over the pulse duration son. Ampli-tude and direction of the radiation force depend on the propertiesof the interrogated material. For small-unbound particles, localradiation pressure gradients can result in particle movement. Inthe special case of standing waves, this can in turn lead to a separa-tion of particles or cells at nodes/antinodes depending on the prop-erties of the particles and fluid. This effect is utilized for example inacoustic tweezers [46]. The assumption that the low frequency ofthe radiation force, but not the pressure alteration at the ultrasoundexcitation frequency, is responsible for the biological effect ob-served in LIPUS, led to studies investigating direct stimulation atthe modulating frequency of 1 kHz, suggesting that the radiationforce is indeed an important component of the stimulation[47,48]. This may also imply that the PRF, i.e. the frequency of theresulting radiation pressure, could have an impact on the biologicaloutcome if cells are sensitive in this range of cyclic loading, anassumption that is supported by recent experimental evidence ob-tained with varying PRF [49].
Attenuation, nonlinear sound propagation (i.e. the conversion ofacoustic energy from the fundamental to harmonic frequencies),and divergence of the acoustic waves in the far-field of the trans-ducer lead to a gradual decrease of the radiation pressure withincreasing distance from the acoustic source. The attenuation inpure water is [50]: a = 2.17 � 10�15�f2(dB/cm), whereas in soft tis-sues it is typically assumed that attenuation is linear with fre-quency and varies as a = 0.3 dB/cm/MHz.
This radiation pressure gradient gives rise to a net force directedaway from the transducer and a resulting fluid flow in liquids(Fig. 2). Solid interfaces hinder this fluid flow induced mass trans-port. However, it has been shown that fluid streaming can build upagain in the acoustic beam after a membrane [45].
Experimentally, it was observed that acoustic streaming (i) in-creases with increasing proportion of nonlinear acoustic propaga-tion, i.e. with the generation of harmonics at higher intensities,(ii) gradually increases with increasing distance from the trans-ducer and (iii) builds up 60% of the free streaming velocity within1 mm after solid interfaces [45]. The streaming velocity in waterand amniotic fluid have been measured at 3.5 MHz (a = 2.82 cm2,focus distance: 95 mm, CW, W = 140 mW, PA = 0.23 MPa,ISATA � 50 mW/cm2) to be in the order of 3 cm s�1 [51]. Muchhigher velocities (up to 9 cm s�1) were observed for high-amplitude short pulse excitation (W = 140 mW, PA = 4 MPa,ISATA � 50 mW/cm2). In cell culture flasks, values of 0.4, 6.0 and19.4 cm/s at 130, 480 and 1770 mW/cm2, respectively, weremeasured at 3 MHz and continuous wave excitation [52]. In thislater study, no cavitation events could be recorded for intensitiesbelow 500 mW/cm2.
Several groups investigated the temperature rise possibly in-duced by LIPUS. They generally ruled out temperature effects, be-cause the measured temperature increase was typically below0.2 �C for intensities (SATP: spatial average, temporal peak) below
m in diameter. The transducer is centered at the origin of the coordinate system inral intensity (ITP) distribution at a distance of 4.4 mm from the transducer surface.
h ultrasound: A review of the possible mechanic effects, Ultrasonics (2014),
Fig. 2. Acoustic radiation force and streaming action on objects in the sound field generated by and acoustic source. (a) In liquids, the radiation force (black arrows) gives riseto acoustic streaming (green arrows). The inhomogeneous intensity distribution in the near field results in a regional dependence of the direction and the amplitude of theradiation force. Acoustic streaming velocity is gradually increasing from the transducer surface to the focus region. Solid interfaces interrupt the flow, but not the radiationpressure. (b) Acoustic streaming, visualized by ten superimposed images taken at 200 ms intervals of corn starch particles in the field of a 32-MHz plane transducer [210].(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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500 mW/cm2 [52] or even 2400 mW/cm2 [44], in experimental set-up where the cell dish was positioned in the focal plane with a spe-cial absorbing chamber, preventing multiple reflections within thedish (see next section). The issue of temperature rise is howeverhighly dependent on the experimental set-up. These effects havebeen studied in detail by Leskinen and Hynynen [53], who reportedmaximum temperature rises of 3 �C at 30 mW/cm2 (ISATA) in exper-imental set-ups in which the transducer was coupled to the bottomof culture plates with coupling gel. In contrast, a maximum eleva-tion of 0.2 �C was observed if the transducer was immersed inwater. This effect is related to the heating of the ultrasound trans-ducer. The poor heat transfer properties of surrounding air leads toa heat transfer through the coupling gel into the well plate. There-fore, temperature-related biological effects may be induced in cellculture systems, in which the transducer is not properly cooled,but not in systems in which the transducer is immersed in a tem-perature controlled water tank.
Besides temperature elevation, radiation pressures, streamingand standing waves effects, propagation of surface shear wavesand Lamb waves have been reported to take place in vitro due tomode conversions at the bottom of plastic wells [53]. These wavescan in turn be responsible for bioeffects and can propagate toneighboring wells, jeopardizing the attempt to precisely controlthe stimulus delivered to a given cell monolayer.
It appears therefore that only cavitation can be ruled out forstimulations intensities below 500 mW/cm2. Mechanical forcesresulting from spatial and temporal gradients of radiation pressureand acoustic streaming, propagation of surface and guided wavesare likely sources for biological effects observed in LIPUS stimula-tion studies. Temperature effects can be ruled out in experimentalset-ups with water coupling, but its potential role in setups usinggel coupling cannot be ignored.
Fig. 3. Various sound exposure set-ups used for in-vitro LIPUS st
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3.3. The influence of geometrical configurations on in-vitro stimulation
For the exposure the ultrasound transducer has to be coupled tothe cell culture dish. Different set-ups have been used, i.e. (i)immersion of the sterilized transducer from the top directly intothe culture medium in close distance (3–5 mm) to the cell layer(Fig. 3a), (ii) direct coupling to the bottom of the culture dish viacoupling gel (Fig. 3b), (iii) exposure from the bottom of the culturedish with the cell layer place in the focal plane and immersion of aspecial sound absorption chamber (Fig. 3c), and (iv) the propaga-tion of the sound through a culture flask (Fig. 3d).
Considering the spatial and temporal sound field dimensions, asillustrated by Fig. 1, and the limited dimension of the culturedishes, several sound propagation phenomena, e.g. inhomoge-neous pressure distribution in the near field, multiple reflectionsat interfaces, standing waves and acoustic streaming have to beconsidered.
The sound propagation distance in the cell culture dish or flask(a few mm) is generally much smaller compared to the totalpropagation distance a wave can travel before it is attenuated.The attenuation in water at 1 MHz and 37 �C is very low(0.0014 dB/cm [54]). If the incoming waves are not properlyabsorbed or directed away from the culture dish, they will bereflected several times at internal interfaces. Due to the long pulseduration of at least 200 ls (which is equal to 300 cycles or in termsof wavelength: 300 � 1 mm = 0.3 m at 1.5 MHz), any interface per-pendicular to the sound propagation direction, e.g. liquid/plastic,liquid/air, or liquid/transducer will result in the development ofstanding waves, ring interferences and a remarkable prolongationof the duty cycle (Fig. 4). As a consequence, the effective peakand time average intensity levels and the resulting modulation ofthe radiation force between ON and OFF phases is considerable dif-
imulation. (a–d) From Refs. [130,124,176,151], respectively.
h ultrasound: A review of the possible mechanic effects, Ultrasonics (2014),
Fig. 4. LIPUS temporal wave pattern in a culture dish with the transducer placed on the bottom of the dish and (a) in presence or (b) absence of a silicon absorbing chamberon the top of the dish (reproduced from Fig. 1 of [211]). Without an absorbing chamber, a gradual increase of intensity during the ON cycle and a gradual decay during the OFFcycle can be seen, which lead to effective slight and remarkable increases of the applied temporal peak (ITP) and temporal average (ITA) intensities, respectively.
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ferent from that measured in an interface-free medium. The esti-mation of the effects of standing waves is not straightforward asthe development of nodes depends on multiple factors [54].
Another important factor is the transmission and reflection ofthe sound waves through the well plate walls. Multiple reflectionsinside the plastic result in pronounced oscillations of the transmit-ted and reflected amplitudes. The resonance frequencies for maxi-mum transmission (fTn) and reflection (fRn) are:
fTn ¼ncd; n ¼ 1;2;3; . . . ; ð8Þ
fRn ¼nc2d
; n ¼ 1;2;3; . . . ð9Þ
whereas c and d are sound velocity and thickness of the plate,respectively.
If the ultrasound frequency is set to the antiresonance value, thetransmitted or reflected wave amplitudes can be completelydiminished [53]. For a typical polystyrene well-chamber with alongitudinal sound velocity of 2305 m/s at 37 �C and a well bottomthickness of 1.22 mm, the transmission and reflection resonancefrequencies are at intervals of 1916 kHz and 958 kHz, respectively.Therefore, stimulations conducted with the same set-up, but dif-ferent frequencies may result in different exposure levels in thewell, if the sound has to be propagated through the well platebottom.
3.4. The LIPUS ‘Dose’ and the need for standardization
Standardization of experimental settings, both in vitro andin vivo, is a crucial factor in order to compare studies, but also tobe able to identify relevant parameters and subsequent physicaland biological effects. This is particularly important in vitro, be-cause both the positioning of the culture plates/wells with respectto the ultrasound source and the output of the source can be re-garded as responsible for a lack of reproducibility of LIPUS treat-ments of cells. The formation of standing waves patterns inparticular have been reported to be very sensitive to the heightof the fluid in the wells and to be responsible for very large spatialvariations of the peak pressure applied on the cells [54,55]. The ef-fects of intensities have indeed been reported to influence the out-come of in-vitro experiments.
The concept of an acoustic ‘dose’ for LIPUS that could be stan-dardized is difficult to derive. Similarly to therapeutic thermalultrasound [56], the dose should be related to the amount of bioef-fect induced. However, given the lack of understanding of whichparameters of the stimulation are responsible for any given bioef-fects, the dose is so far a lacking concept in the field of LIPUS. Wetherefore suggest that precise report of transducer geometry,intensity, peak pressure, duty cycle, pulse duration, center
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frequency be reported, and, especially for the in-vitro case, greatcare be given to the geometry of the experimental set-up, to eval-uate the aforementioned parameters in the well, and in particularto avoid the formation of standing wave patterns and heating bythe transducer. For historical reasons, most of the studies havebeen performed using parameters similar to the Exogen commer-cial system, i.e.: plane transducer, ISATA = 30 mW/cm2, 200 lspulses, 1 kHz PRF, treatment time 20 min daily. Unless otherwisementioned, our discussion is focused on data obtained using simi-lar settings.
4. Potential bio-effects of LIPUS
The difficulty in interpreting LIPUS experiments lies in the com-plexity of the triggered physical phenomena that can in turnpotentially induce induced bio-effects. Compared to experimentaldesigns implying laser traps, controlled fluid shear stress or twist-ing of magnetic beads, it is difficult to isolate a single pattern ofmechanical stimulation by a propagating ultrasound wave. Thisproblem is even more severe, as several of these acoustical poten-tial phenomena can be activated with similar acoustic outputparameters to a variable extent, depending on the particular geom-etry of the stimulation system, [54] and that the biological re-sponse to the treatment may depend on cell and tissue types [1].
Potential LIPUS physical effects that may induce a biological re-sponse can be divided into thermal and non-thermal effects.
4.1. Thermal effects
Temperature rises, associated with LIPUS, can range from a fewtenth of degrees to a few degrees at the bottom of the culture wellwhen coupling gel is used [53]. These changes can be significantenough to regulate thermo-sensitive enzymes like metalloprotein-ase, which support 3-fold reaction rate increase per each 2 �C raiseand are important enzymes for bone matrix remodeling [57,58].These associated effects should be further investigated. Forwater-coupling experimental setups, it is a fair assumption to con-sider that thermal effects are not determining, but rather additiveto the non-thermal effects.
4.2. Non-thermal effects
Non-thermal effects imply dynamic mechanical forces at variousfrequencies (from quasi-stationary up to the acoustic excitation fre-quency) [59,47,48]. A bulk ultrasonic wave is a propagatingmechanical perturbation of the medium, and for a frequency of1 MHz (typical center frequency for LIPUS devices) the correspond-ing wavelength is about 1.5 mm in soft tissues or in culture med-ium. This wave can directly affect the mechanosensitive elements
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(see next section) by inducing oscillatory strains at very high fre-quency (typically thousands to a few millions oscillations per sec-onds) compared to physiological strains. The strain amplitudeswill be function of the applied intensity, and are typically of the or-der of 10�5 for low intensity ultrasound. Secondary mechanisms atlower frequencies can also be triggered by the acoustic radiationforce [59] – an oscillatory strain acting at the frequency correspond-ing to the pulse repetition frequency for pulsed ultrasound (typi-cally 1 kHz for LIPUS-type stimulation), resulting in a lowfrequency cyclic mechanical stimulus [47,60]. Inhomogeneities inthe acoustic near field, or caused standing waves and ring interfer-ences, can also generate local strain gradients. Finally, fluid-flow re-lated phenomena can also be induced, such as acoustic streamingresulting in a quasi-stationary fluid flow [51], and microstreaming,i.e. fluid streaming around oscillating particles.
The relevant scale at which these effects will influence the bio-logical response on the cells or tissues in the context of bone heal-ing, remain an open question.
4.2.1. Effects at the tissue and cellular scalesRadiation force, fluid flow and strain gradients can create shear
stresses on cell membranes. Acoustic streaming and microstrea-ming, which are often used in the literature interchangeably,although consequences of two separate physical phenomena, arethought to play important roles in LIPUS action; especiallyin vitro. The former is associated with a transfer of momentum tothe fluid, which will result in nutrients redistribution in the culturemedium in vitro and perturbation of the local homeostasis, trigger-ing biological responses. The latter is generated in response tooscillating gas bubbles or other small acoustic inhomogeneities,causing circulatory movement of the fluid. Microstreaming aroundgas bubbles in response to LIPUS is not likely to happen in vivo inbone tissue or in the fracture callus, because the pressure levels ap-plied with LIPUS systems are well below cavitation thresholds.However, it can be hypothesized that the wave propagationthrough the highly porous network of soft and mineralized tissuesin the fracture callus may induce fluid micromovement in thepores. Fluid flow, created by either acoustic streaming or micros-treaming, can modulate the extracellular matrix and can applyshear stress activating mechanoreceptors on the cell membranes.
4.2.2. Intracellular effectsDirect mechanical action of the ultrasound wave on cell mem-
branes or proteins may trigger a biological response. At intensitiesbetween 1 and 2 W/cm2 (i.e. much higher than applied by typicalLIPUS) the very low strains induced by ultrasound on cellsin vitro have been reported to induce a prompt fluidization of thecytoskeleton resulting in accelerated cytoskeletal remodelingevents [61]. These mechanisms, similar to those caused by physio-logical strains of higher amplitude and much lower frequency,could contribute to the bioeffects of LIPUS.
Other proposed mechanisms of action of pulsed ultrasound atthe intracellular scale include the hypothesis of relative oscillatorydisplacements between intracellular elements of different densi-ties [62], such as cell nucleus, and the structure in which theyare embedded. By modeling the linear oscillations of such struc-tures with different rheological models, Or and Kimmel [62] sug-gested that LIPUS can trigger cyclic intracellular displacementscomparable with, and even larger than, the mean thermal fluctua-tions, with resonance frequencies in the range of tens to hundredsof kHz, so within the range of direct oscillatory movement causedby the ultrasound wave or by the acoustic radiation pressure. Addi-tionally, the ‘bilayer sonophore’ model [63] they proposed, de-scribes a mechanism where the intramembranous hydrophobicspace between the two lipid monolayer leaflets inflates and defla-tes periodically when exposed to ultrasound, being pulled apart
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during the depression phase (negative pressure) of an ultrasoundcycle, and pushed back together by the positive pressure. The mainassumption being that negative acoustic pressures are large en-ough to overcome molecular attractive forces of the two leafletsof the cell membrane. Published data showed that this effect canoccur at levels below cavitation thresholds, but were obtained withintensity levels higher than typically used in LIPUS treatment(1 W/cm2 at 1 MHz). Nevertheless, this is an interesting model thatwould merit further investigation with LIPUS-type ultrasoundfields.
Another mathematical model was proposed to predict ultra-sound-induced intra-cellular stresses and strain, using a biphasic(solid elastic structures and macromolecules on one side, fluidcytosol on the other side) description of the cells in a harmonicstanding wave field [64]. Modeling results predict that stressesand strain are maximized within the cell at two distinct resonantfrequencies, which are cell-type dependent through the geometricand mechanical characteristics of its different components. Stres-ses gradients are induced though dilatational deformation, result-ing in net force acting on the nucleus, possibly triggeringtransduction by the nucleus and leading to load-inducible geneexpression. Stimulated load-inducible gene expression shouldtherefore be maximized when excitation frequency matches thecell resonant frequency, a prediction that was confirmed by exper-imental data [64].
4.2.3. Molecular effectsAt the molecular scale, it is well known that ultrasound can
interact with molecules. This interaction is used in the field of‘‘molecular acoustics’’ to probe molecular properties, based onthe measurement of variations in ultrasound velocity [65] orabsorption [66]. These measurements utilize the changes in molec-ular compressibility (and in ultrasound velocity) or in ultrasoundabsorption following changes in conformation, in solvent–mole-cule interactions, or in hydration. All these effects are the resultsof intra and intermolecular forces, interaction potentials, electro-static interactions, rigidities of interatomic bonds and relaxationphenomena. Lipids for example, constituting the second most pre-valent component of dry weight, after proteins, of many tissues,interact with ultrasound. Lipids and structures associated withthe cell membranes exhibit an absorption behavior for ultrasound,probably linked to relaxation phenomena, with relaxation frequen-cies in the range 1–16 MHz. In large unilamellar vehicles, theserelaxation phenomena are linked to the interaction of ultrasoundwith the hydrophobic side chains, leading to a structural reorgani-zation of small domains of molecules [66]. These interactions canaffect the function of biological molecules. At very high intensities,ultrasound can damage biological molecules, e.g. to degrade DNAin the presence [67] or in the absence of cavitation [68], but theseintensities are much higher than those typically used in Low Inten-sity Pulsed Ultrasound stimulation or other techniques mentionedin this review (for an in-vivo use of ultrasound, safety guidelinesare provided by scientific societies or regulatory agencies, in orderto avoid such effects). At lower intensities however, where ultra-sound interaction with molecules can be observed as translatedby absorption, can the deposited ultrasound energy induce tran-sient conformational changes or changes in molecules–moleculesor molecules–solvent interactions that in turn would modify thebiological functions of these proteins? It has been suggested thatultrasound could disrupt multimolecular complexes [69], althoughintensities and mechanisms (cavitation or not) at which these phe-nomena could occur were not specified. A change of protein con-formation is believed to be responsible for fluidization of thecytoskeleton observed after low intensity therapeutic ultrasoundtreatment at 1 MHz center frequency, with 20% duty cycle andpressures of the order of 200 kPa [61]. The origin of these phenom-
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ena is thought to be due to the local strains applied by ultrasoundthat could be large enough to disrupt weak nonspecific bonds,altering protein conformation and triggering structural remodelingof the cytoskeleton.
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4.2.4. Other effectsFinally some reported ultrasound bioeffects are difficult to cate-
gorize due to the lack of knowledge of the physics underlying them.In particular, ultrasound can affect intracellular trafficking, and thiswould be relevant in the stimulation of the bone healing processes.It has been recently reported that after a few minutes of exposure to1 MHz ultrasound (at ISATA of 0.3 W/cm2, 50% duty cycle and 5 HzPRF), cells that had already internalized plasmids (delivered usingcalcium phosphate co-precipitates) expressed higher transfectionrates than the non-ultrasound treated cells [70]. The mechanismsof these effects are not clearly understood, but may be related toeither a destabilization of the endosomal vesicles, or through an in-crease of the net diffusivity of pDNA through the cytoplasm, aninhibitory action on enzymatic level, or assistance in the permeabi-lization of the nuclear membrane. Whatever the exact mechanismsbe, this study clearly demonstrated that ultrasound at levels belowcavitation thresholds can modulate intracellular trafficking ofpDNA, and by extension could more generally modulate intracellu-lar trafficking of other molecules, thereby eventually triggering orenhancing a possible mechanoresponse.
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5. LIPUS and mechanotransduction
Conventionally, ‘mechanotransduction’ is the process in whichspecific cellular machineries switch a physical stimulus into chem-ical activities to trigger downstream signaling. Conformationalchanges in proteins, such as stretch-activated ion channels ormechanosensitive adhesion structures, often mediate conversionof force into chemical signaling. Given the modalities of mechani-cal stimulation of the LIPUS as exposed in Section 4, it is difficult atthis stage to specify a biological response to a particular strainrange. Nevertheless, the magnitude of interfragmentary move-ment, the rate and timing of application of micromotion and thenumber of loading cycles are mechanical boundaries susceptibleto impact signal transduction pathways and subsequent genesup- or down-regulated, thereby influencing cell phenotype andfunction. A variety of mechanosensitive membrane moleculesand microdomains have been identified, including ion channels,receptors, G-proteins, adhesion molecules, caveolae, the glycoca-lyx, and primary cilia. As the intracellular cytoskeleton ultimatelybears the impact of force application to the cells, it also representsa major class of mechanosensitive structures. The activation, rein-forcement and/or redistribution of membrane-bound complexes,and the reorganization of the cytoskeleton and extracellular ma-trix, are all immediate responses to forces. These events criticallyinfluence cell metabolism, protein synthesis, cell survival, in addi-tion to migration, spreading and contraction. As compared to othertypes of mechanical stimuli (such as shear stress or extracellulardeformation), ultrasound-induced mechanotransduction pathwaysare less well defined. We will present mechanosensitive structuresthat potentially should come into play knowing that the sensormolecules that initially sense the different varieties and orders ofmagnitude of mechanical stimuli are not fully known.
Changes of intracellular free calcium concentration are one ofthe first responses against environmental stress and are importantbiological signals in mechanotransduction: Upon ion channels acti-vation, calcium is mobilized into cytoplasm from outside of the cellmembrane or from intracellular reservoirs including endoplasmicreticulum, sarcoplasmic reticulum and mitochondria. There areexperimental evidences suggesting that ultrasounds, even at low
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intensity, increase the intracellular concentration of calcium [71]and that blocking this rise with intracellular calcium chelatingagents, or inhibiting Ca2+/ATP-ase, abolishes the stimulatory effectof ultrasound on proteoglycan synthesis by chondrocytes. Prolifer-ative LIPUS effects were also shown to depend on the release ofATP/purines through Ca2+ and the P2Y receptors [72]. Calcium isa product generated indirectly from enzymatic activities like phos-pholipase C. In consequence of the intracellular calcium increase,calcium acts as a secondary messenger by allosterically bindingto proteins including calmodulin, troponin-C, annexin as well ascalpain and then the complex triggers downstream cellular pro-cesses. Recently, it has been shown that Ca2+ signals are oscillatory[73] and that these oscillatory Ca2+ signals are crucial for a varietyof cellular functions such as bone marrow-derived mesenchymalstem cells (HMSCs) differentiation [74,75]. In HMSCs, calciumoscillation can be regulated by electrical stimulation [76] or sub-strate rigidity [73]. In this later study Ca2+ oscillation occurredvia RhoA GTPases pathway, who are major regulators of cytoskel-eton, although sometimes not correlated with cytoskeleton activi-ties [77]. However at this stage we do not have evidence thatoscillatory Ca2+ signals are a target for LIPUS stimulation, and fur-ther studies should address whether intracellular Calcium rise is aprimary target or a downstream event.
Another major actor in mechanotransduction of adherent cells,being at the center of cell integration of internal and external sig-nals, is the ‘‘cytoskeleton – focal adhesions – extracellular matrix(ECM) connection’’. Integrin-mediated focal adhesions (FAs) arelarge, multi-protein complexes that link the actin cytoskeleton tothe ECM and take part in adhesion-mediated signaling. FAs insurecell adhesion to ECM via integrin-regulated organization. They un-dergo maturation wherein they grow and change composition dif-ferentially to provide traction and to transduce the signals thatdrive cell migration, which is crucial to various biological pro-cesses, including wound healing. FAs are related to signaling net-works and dynamically modulate the strength of the linkagebetween integrin and actin and control the organization of the ac-tin cytoskeleton [78,79]. In response to mechanical force, theseforce-sensitive focal adhesion proteins may undergo structuralrearrangement or enzymatic modifications that change their bind-ing preferences with respect to other associated proteins and thisthen further modulates the protein association with focal com-plexes [80]. It has been shown that forces in the physiologicalrange (2-20pN) are sufficient to stretch FA molecule (talin), expos-ing cryptic binding sites for others molecules (vinculin), suggestinga role for protein binding in the talin-vinculin system in the mech-anotransduction process. LIPUS-type mechanical stimulation hasbeen shown to alter the role of integrins [81]. It was shown to acti-vate the integrin/phosphatidylinositol 3-OH kinase/Akt pathwaywhich is associated with cell proliferation [82]. It was also shownto trigger phosphorylation of FAs signaling molecules such asFAK, Src, p130Cas, which in turn phosphorylate and activate Erkand MAP Kinases. LIPUS signal transduction to the nucleus viathe integrin/MAPK pathway influences numerous cellular pro-cesses including migration and a wide range of gene regulation[81]. Among genes whose expression is promoted by LIPUS arechemokines as monocyte chemoattractant protein (MCP)-1, mac-rophage-inflammatory protein (MIP)-1 and RANKL in osteoblasticcells [83], TNF-a in macrophagic cells [84].
ECM compounds were also stimulated by LIPUS exposure: typeIX-collagen in chondrocytes in 3D matrices [82], proteoglycan andgrowth factors and their receptors (BMP2, FGF7TGF-b R, EGFRF1,VEGF) in nucleus pulposus cell line [85]. In the osteosarcoma cellline, UMR-106, 20-min exposure to LIPUS up-regulated mRNA lev-els for alkaline phosphatase and osteocalcin suggesting a beneficialeffect of LIPUS on bone formation (implication of these results arediscussed in Section 6).
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It is recognized that the anabolic biophysical effects caused byLIPUS are most likely to be caused by mechanical stress and/orfluid micro-streaming impacting on the cellular plasma mem-brane, focal adhesion and cytoskeletal structures to trigger intra-cellular signal transduction and subsequent gene transcription.However other potential candidates should be investigated suchas stretch activated ion channels or G-protein coupled receptors[86], the glycocalyx, a pericelllular GAG-proteoglycan rich layersurrounding the cell membrane that creates a drag force whenfluid flow passes over causing plasma membrane deformation[87], and the primary cilium, an immotile microtubule-based orga-nelle, that protrudes like an antenna from the apical cell surface,and that has been implicated as a mechanosensor in a variety ofcell types including MSCs [88].
Along with transmembrane integrins, direct cell-to-cell com-munication is important for mechanotransduction. The connexinsnot only experience the different biomechanical forces within thesystem but also act as effector proteins in coordinating responseswithin groups of cells towards these forces. Improved cell-to-cellcommunication via gap junctions was reported after LIPUS expo-sure in rat MSCs, which was assessed by dye transfer assay. Inhibi-tion of gap junctions led to decreased activation of Erk1/2 and p38MAPK kinases and attenuated ALP activity in response to LIPUS,implying that gap junctions are essential for LIPUS effect on osteo-genic differentiation of MSCs [89]. Activation of p38 and Erk1/2MAPK kinases in rat MSCs and Erk1/2 MAPK kinase in murinepre-osteoblastic MC3T3-E1 cells have been confirmed by otherinvestigators [90,83].
Activation of another mechanoreceptor on the surface of osteo-blasts, angiotensin II type I receptor (ATI), has been suggested byBandow et al. [83]. The ATI receptor expression has increased withmaturation of osteoblasts and inhibition of the receptor resulted inablation of LIPUS-induced cytokine expression and Erk1/2 phos-phorylation. To date, integrins remain the most-studied receptors,known to transmit US induced signals into the cells. The existenceof other mechanoreceptors, like ATI, mechanofunction of which isnot well understood, as well as more detailed functioning of signal-ing pathways transmitting the converted mechanical stimuli intra-cellularly should be further investigated.
Further how LIPUSs-induced cues, from the nanoscale-level tothe tissue-level, compromise tensional homeostasis still need tobe investigated to provide a comprehensive understanding of coor-dinated mechanoresponsiveness.
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6. LIPUS induced bone healing: biological evidences
Bone healing is a complex biological phenomenon, composed ofa temporally and spatially overlapping sequence of four basicstages: inflammation, soft callus formation, bone formation andbone remodeling, that to some extent recapitulates the develop-ment bone formation processes [91,92]. LIPUS has been reportedto actively influence all these stages, i.e. (1) to mitigate the soft tis-sues phases (inflammation and soft callus formation), (2) to poten-tially accelerate the onset of bone formation and (3) to influencethe biomechanical properties of the remodeled bone [93]. Thereare various ways by which LIPUS might play a role in these thisorchestrated sequence of events, including affecting migration,proliferation, differentiation of several cell types (e.g. mesenchy-mal stem cells, osteoblasts, chondrocytes, osteoclasts, fibroblasts,endothelial cells, and inflammatory cells), ECM production, andremodeling. In particular, mesenchymal stem cells (MSCs), pos-sessing multiple-lineage differentiating potential, can form cellsfrom bone, cartilage, fat, muscle, tendon, etc. and, therefore, receiv-ing more and more attention in the field of regeneration therapies[94].
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Numerous phenomenological studies have investigated cellularevents in response to LIPUS exposure. Fig. 5 illustrates possibleultrasound effects on the sequence of events in healing bone frac-ture, summarizing gene expression and release of chemical mes-sengers in response to ultrasound as determined by in-vitrostudies, which are described in details in Section 6.1. Furthermore,signaling pathways important for transduction of mechanostimuli,generated by LIPUS are discussed in Section 6.2 and summarized inTable 1. At last, the possible role of ultrasound in maintenance ofextracellular fluids homeostasis is stipulated in Section 6.3.
6.1. LIPUS-stimulated gene expression and signaling molecule release
Fig. 5 depicts the effect of ultrasound on each stage of fracturehealing. The data have been gathered from in-vitro reports andare presented in the figure within 4 distinct phases correspondingto the four basic stages of the bone healing process. Phase 1 illus-trates the possible early effects of LIPUS, observed soon after thebone injury, including hematoma formation, inflammation andmigration of precursor cells. Phase 2 shows LIPUS-effect on angio-genesis, MSCs proliferation, osteoblasts proliferation and differen-tiation. Phase 3 depicts LIPUS-enhanced chondrogenesis andosteoblasts maturation. Phase 4 represents further differentiationof chondrocytes, LIPUS-accelerated woven bone formation andremodeling. The information gathered in figure and in Table 1 doesnot recapitulate all the complexity of biological mechanisms in re-sponse to ultrasound treatment, but rather represents an attemptto reconstitute a hypothetical consequence of cellular eventswhich were observed during in-vitro studies, performed in variouscell types and species. We will now review these data in moredetails.
6.1.1. InflammationSoon after the bone fracture, fibroblasts form a granulation tis-
sue to support the damaged site and a hematoma is formed. Theinfluence of ultrasound stimulation on proliferation of fibroblastswithin 24 h post-exposure has been reported by several studies[95–97].
Hematoma formation is accompanied by the release of chemo-tactic factors recruiting inflammatory cells, MSCs and other pro-genitors to the fracture site. Kumagai et al. [98] reportedrecruitment of local osteoprogenitors and of osteogneic precurorsof systemic circulation to the fracture site in mice in-vivo modelafter LIPUS treatment (Fig. 5, ph.1).
6.1.2. AngiogenesisIt has been shown that LIPUS up-regulates interleukin-8 (IL-8)
secretion by human mandibular osteoblasts [96] and gene expres-sion in murine pre-osteoblasts [83] (Fig. 5, ph.1). IL-8 is a cytokineknown to induce endothelial cells proliferation and migration,which, in turn, are crucial for new blood vessels formation (angio-genesis) in the healing of fracture [99,100] (Fig. 5, ph. 2).
Enhanced production of vascular endothelial growth factor(VEGF) by human mandibular osteoblasts, human peripheral bloodmonocytes [96] and human osteoblasts (hFOB1.19, MG-63 andSaOS-2) [101] has also been reported in response to LIPUS (Fig. 5,ph.1). VEGF is a critical player in angiogenesis. It regulates mito-genesis and the recruitment of endothelial cells [102], and is in-volved in osteoblasts differentiation and osteoclasts activation[103,104] (Fig. 5, ph. 2). Basic fibroblast growth factor (bFGF), an-other cytokine regulating angiogenesis in the healing fracture[105,106] has been found to be up-regulated by LIPUS treatmentin human mandibular osteoblasts [96]. bFGF enhances fracturehealing through an unknown mechanism by promoting migration(Fig. 5, ph.1) and mitogenesis of MSCs and osteoblasts [107–109](Fig. 5, ph.2).
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Fig. 5. Summary of hypothetical LIPUS effects on cellular events from published in-vitro data. The columns represent the four phases during in-vivo endochondral bonefracture healing: phase 1 – early events soon after the bone injury, i.e. hematoma formation, inflammation and migration of osteogenic precursors; phase 2 – angiogenesis,proliferation of MSCs and osteoblasts and osteogenic differentiaion; phase 3 – chondrogenesis and maturation of osteoblasts; phase 4 – maturation of chondrocytes, wovenbone formation and remodeling.
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Co-cultures of human osteoblasts SaOS-2 and human umbilicalvein endothelial cells exhibited higher levels of platelet-derivedgrowth factor (PDGF) secretion upon LIPUS exposure [110–112].PDGF is a mitogenic factor for MSCs and osteoblasts [110–112](Fig. 5, ph.2).
6.1.3. NO and PGE2Nitric oxide (NO), a free radical gas, was found to be an impor-
tant contributor to bone formation in response to mechanical load-ing [113]. The up-regulation of NO production in response to LIPUSwas demonstrated to be involved in the regulation of VEGF expres-sion in human osteoblasts (hFOB1.19) [101] (Fig. 5, ph.1). Theincrease of NO production is in agreement with findings of Reher
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et al. [42], who also showed that NO concentration in human pri-mary mandibular osteoblasts increases using both continuousand pulsed ultrasound systems and established that the increasewas regulated by inducible NO synthase (iNOS) (Fig. 5, ph.1).
Secretion of prostaglandin E2 (PGE2) has been shown to be up-regulated in response to ultrasound exposure in osteoblasts fromvarious species [42,114–116]. PGE2 is an arachidonic acid-derivedmetabolite, associated with bone formation and resorption [117].Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme in reactionof PGE2 production. It has been reported that LIPUS increasesCOX-2 gene expression in mouse pre-osteoblasts [118] (Fig. 5,ph.1). In cells, pretreated with a COX-2 specific inhibitor, PGE2 pro-duction in response to ultrasound was significantly decreased.
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20 min 30 min post-exposure PKCa, mediating Rac activity in thepresence of syndecan-4, was notinvolved in US-induced activity of theprotein
[114] p-FAK " 1.5 MHz, 30 mW/cm2, 200 lsburst at 1 kHz PRF transducer isfrom the top, 5–6 mm away fromthe cells
MC3T3-E1mouse pre-osteoblasts,primaryosteoblasts
20 min Peak after 10-30 min The activation of the listed pathways isimplicated with enhanced expression ofCOX-2, which is supported by highersecretion of PGE2 in the medium
p-p85 of PI3K "p-Erk1/2 "p-Akt " activation ofcanonical NF-jBpathwayPGE2 " 6 h post-exposure,
Inhibition of p-38 reduces US-increasedALP activity, OC secretion and matrixmineralization
(not clear how it is pulsed)
[163] p-Erk1/2 " 1.5 MHz, 70 mW/cm2, 2 ms burstat 100 Hz PRF, transducer is fromthe top, 3-4 mm away from thecells
C2C12 mouse 20 min 60 min post-exposurestarting at 10 min andpeaking at 30 min
The pathways drive differentiation ofC2C12 cells into chondroblastic/osteoblastic lineages
p-p38 "
p-p38 " 10 min post-exposure[81] p-FAK (Y397) " 5 MHz in continuous mode,
Spatial averaged pressure 14 kPa,transducer immersed in themedia 6 mm away from thebottom
human primarychondrocytes
3 min 60 min post-exposure Integrins and Src inhibitors lowered US-induced phosphorylation of Erk1/2,implying that they function upstreamfrom the protein
p-Src (Y416) "p-p130Cas (Y249) " 5 min post-exposure
and decrease by 60 minp-CrkII (Y221) "p-Erk1/2 "
[82] p-Akt " 1.5 MHz, 30 mW/cm2(temporalaverage), 200 ls burst at 1 kHz,PRF, transducer is from thebottom via coupling gel
Primary pigchondrocytes in3D collagenmatrix
20 min/dayfor 7 days
2 h post-exposure onday 7
Akt pathway could be involved in US-increased chondrocytes proliferation,which was confirmed by up-regulationof Cyclins D1 and B1
Transient increase at 111 kPa andsustained rise at 450 kPaIncreased [Ca2+] was established to beimplicated with US-inducedproteoglycan synthesis
[89] p-Erk1/2 " 1.5 MHz, ISATA = 30 mW/cm2,200 ls burst at 1 kHz
Rat bonemarrow stromalcells
20 min 30 min post-exposure The inhibition of gap-junctions abolishesUS-enhanced phosphorylation of the p-Erk1/2 and p38p-p38 " Tranducer is from the bottom via
cm2, 200 ls burst at 1 kHz PRF.Rat bonemarrow stromalcells (inosteogenicmedia)
20 min 30 min post-exposure More intense phosphorylation of p-p38is seen already at 15 mW/cm2, whereasp-Erk1/2 phosphorylation is only morepronounced at 30 mW/cm2
p-p38 "
[83] p-Erk1/2 " 1.5 MHz, ISATA = 30 mW/cm2,200 ls burst at 1 kHz PRF,transducer is 13 cm away fromthe bottom in the water tank
MC3T3-E1(3 weeksdifferentiated)
20 min 5 min post-exposure AT1 receptor inhibitor down-regulatesLIPUS-induced enhanced Erk1/2activation
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6.1.4. Early osteogenesisInsulin-like growth factor-1 (IGF-1) message has been reported
to be up-regulated by LIPUS in both murine ST2 bone marrow de-rived cell line and rat osteoblasts within the first day of the stim-ulation [116,119] (Fig. 5, ph.1). IGF-1 mediates expression ofosterix (Osx), a transcription factor involved in differentiation ofpre-osteoblasts in mature osteoblasts (Fig. 5, ph.2) [120,121]. Theup-regulated expression of Osx has also been shown by Suzukiet al. [122] in rat osteosarcoma-derived cell line expressing osteo-blastic phenotype, starting on day 7 of LIPUS exposure persistinguntil the end of week 2 (Fig. 5, ph.2,3).
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The up-regulation of early response gene c-fos have beenreported in rat MSCs, rat UMR-106 osteoblast-like cells, primaryrat osteoblasts and murine ST2 cells, after the ultrasoundtreatment [116,119,123–125] (Fig. 5, ph.1). Enhanced expressionof c-jun, c-myc and Egr-1 genes in rat MSCs 3 h after 20 min LI-PUS-stimulation has been demonstrated by Sena et al. [124](Fig. 5, ph.1). The genes c-myc and Egr-1 encode for transcriptionalfactors, involved in osteoblasts proliferation and differentiation[126–128]. The combination of c-jun and c-fos proteins forms anactivator protein-1 (AP-1) transcriptional factor complex. AP-1binding sites are encountered in several promoters, which are
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important for the expression of osteogenic markers, i.e. collagentype 1 (Col-1) and osteocalcin (OC) [129]. The early up-regulationof these proteins as well as other important osteogenic markers,like osteonectin (ON), osteopontin (OPN), bone sialoprotein (BSP),alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2) in cells with osteoblastic phenotype within 1 day followingthe ultrasound-exposure have been demonstrated [116,119,130–132] (Fig. 5, ph.1).
6.1.5. Proliferation of osteoprogenitors and osteoblastsProliferation of osteogenic progenitors is not directly affected
by LIPUS exposure in vitro, as reported by Hasegawa et al., whofound that number of human hematoma-derived progenitor cellsremained the same within 7 days of stimulation, whereas differen-tiation of the cells was significantly enhanced within 28 days of LI-PUS-exposure [133]. LIPUS effect on proliferation of osteoblastsappears to be controversial. Doan et al. [96] and Reher et al. [97]demonstrated that DNA synthesis in human mandibular osteo-blasts was increased 24 h post-exposure (Fig. 5, ph.1). Haytonet al. [134] showed an increase in gene expression of ornithinedecarboxylase (ODC), a cell growth marker, 8 hours later the LI-PUS-treatment in human SaOS-2 osteosarcoma cell line (Fig. 5ph.2). These results are in agreement with a study by Wang et al.[101], where increased DNA synthesis was observed 24-hourspost-ultrasound exposure in cultures of human osteoblastshFOB1.19, MG-63 and SaOS-2. Several more studies reported anelevated proliferation rate in cells with osteoblastic phenotypewithin 4 days of LIPUS stimulation [122,130,135,136,134]. In con-trast to these findings, Suzuki et al. and Dalla-Bona et al. did notsee any change in proliferation of rat ROS 17/2.8 osteosarcomacells and mouse OCCM-30 cementoblast cell line respectively,upon ultrasound-stimulation. Sawai et al. looked at proliferationof mouse osteoblasts MC3T3-E1 and osteosarcoma (mouse LM8and human SaOS-2), renal cancer, prostate cancer and lung cancercell lines and found that it was not affected by LIPUS during 3 daysof stimulation, arguing for beneficial effects of ultrasound usage inthe patients with bone cancers and bone metastases. Increasedproliferation of murine primary calvarial osteoblasts reported inGleizal et al. study [130], was accompanied by enhanced expres-sion of osteogenic markers, i.e. ALP, BMP-2, BMP-7, OPN, Col-1,etc. Expression of these markers was normalized to expression ofa house-keeping gene, compensating for a potential increased cellnumber. Therefore, it is not yet completely clear whether the ben-eficial effects of LIPUS to the fast and favorable healing of the frac-ture observed in vivo are due to enhanced differentiation ofosteoblasts or to a more complex combination of accelerated pro-liferation, differentiation, and maturation, which finally leads to anincreased tissue size.
6.1.6. OssificationAnother transcriptional factor with an essential role in the
development of osteogenic phenotype is Runx2. It has been re-ported that Runx2 can be up-regulated following ultrasound stim-ulation. Up-regulation can start from day 2 following ultrasound inosteoblastic cell line ROS 17/2.8 and in rat MSCs [122,135], and canlast up to 2 weeks following ultrasound in mouse calvarial primaryosteoblasts [122,130,135]. Runx2 is encoded by the Cbfa1 gene(core-binding factor-1) and is only found in cells of osteoblast line-age [137]. Mice with Cbfa1�/� homozygous mutation are not capa-ble of either intramembranous or endochondral ossification[138,139]. Runx2 is a transcription factor regulating differentiationof MSCs into osteogenic lineage by directing expression of ALP, Col-1, matrix metalloproteinase-13 (MMP-13), BSP, OPN and OC genes[137]. Enhanced expressions of ALP and MMP13 have been shownon day 10 and elevated ALP activity was detected on day 6 inLIPUS-treated murine MC3T3-E1 cells [140] (Fig. 5, ph.3).
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Up-regulated OPN and ALP expressions by ultrasound-exposureon day 3 were also shown in rat MSCs and osteoblasts like cells[119,122,130,132,135,140].
An effect of ultrasound on Dlx5 and Msx2 gene expression hasbeen reported by Suzuki et al. [122]. Increased message of bothDlx5 and Msx2 was observed within the first 2 weeks of LIPUStreatment in rat osteoblasts-like cells. The functioning mechanismsof these proteins are not entirely understood. However, it is knownthat Dlx5 gene is important for osteoblasts differentiation and mat-uration, whereas Msx2 is expressed the most during osteoblastproliferation and it antagonizes Runx2 [141]. Dlx5 in turn bindsto Msx2 and, therefore, rescues a prominent transcriptional activ-ity of Runx2 [141].
6.1.7. BMPsBone morphogenetic proteins (BMPs), cytokines of transform-
ing growth factor-b (TGF-b) superfamily, perform a plethora offunctions throughout diverse tissues, regulating cellular eventsbased on the context [142].
BMP-2, 4 and 7 are important players in bone healing and reg-ulate the differentiation of MSCs to the osteoblastic and chondrob-lastic lineages [143,144]. BMPs signal through Smad, a family oftranscription factors, and other cascades including mitogen-acti-vated protein kinase (MAPK) pathways like p38 and Erk1/2[145,146].
These proteins, as well as BMP receptors BMPR-I, II and activinreceptors ACVR-I, II were found to be up-regulated after 7 days oftreatment with LIPUS in rat osteosarcoma ROS 17/2.8 cell line[147]. Enhanced expression of BMP-2 from day 5 until day 14has also been shown in these cells [122]. Smad1 activation, a sig-naling molecule downstream from BMPR receptors, was observed5 minutes after LIPUS exposure.
6.1.8. ChondrogenesisDuring the third stage of bone repair, proliferating MSCs start
differentiating into chondrocytes and endochondral ossificationbegins (Fig. 5, ph.3). Lee et al. reported that low intensity ultra-sound delivered in a continuous-wave mode promoted enhancedexpression of chondrogenic markers Col-2, aggrecan (proteoglycan(PG)), Sox-9 (a gene encoding for a transcriptional factor, promi-nent for chondrocyte differentiation) at week 1 and 2 in rabbitMSCs [148] (Fig. 5, ph.3). Schumann et al. [149] demonstrated thathMSCs seeded in 3D scaffold and stimulated with 40 min a day LI-PUS for one week and then left in the incubator for another twoweeks, have more pronounced chondrogenic phenotype on day21, expressing higher aggrecan, Col-1, Col-2 and Col-10 (Fig. 5,ph.4). Other studies showed that LIPUS supported differentiationof rat and pig primary chondrocytes within the first two weeksof the treatment and up-regulated expressions of TGF-b, TGF-bR1, PG, Col-2 and Col-9 [82,85,150] (Fig. 5, ph.3). Parvizi et al.[71] reported that proteoglycan increase in rat chondrocytes trea-ted by ultrasound was governed by increase of intracellular Ca2+
concentration.Not only differentiation state, but also viability of chondrocytes
is affected by LIPUS. Several studies have shown that proliferationof chondrocytes from rat, pig and human nucleus pulposus cell line(HNPSV-1) was accelerated within two weeks of LIPUS treatment[82,85,150] (Fig. 5, ph.3).
6.1.9. Bone remodelingAt the primary spongiosa level, chondrocytes become hypertro-
phic and begin to secrete ALP, which initiates the matrix mineraliza-tion. The mineralized cartilage matrix provides a scaffold formigration of osteoprogenitor cells, which then differentiate and pro-duce osteoid, the organic matrix of mineralized bone tissue (Fig. 5,ph.4). Enhanced expression of maturating osteoblasts markers, like
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OC, BSP, and calcium deposition in response to ultrasound has beenconfirmed throughout several studies [90,122,131,140,151,152].Receptor activator of NF-jB ligand (RANKL) and osteoprotegerin(OPG) proteins, expressed by osteoblasts, are responsible for regula-tion of osteoclasts functioning. While RANKL activates osteoclasts[153], OPG antagonizes its action by binding to the receptor activa-tor NF-jB (RANK) expressed on the osteoclasts [154]. The most pro-nounced peak (about 10 fold compared to unstimulated controls) ofRANKL gene expression was found during the third week of LIPUS-treatment in differentiating murine osteoblasts, while it was onlyslightly up-regulated during 1st, 2nd, and 4th week of stimulation,whereas OPG expression levels remained constant throughout3 weeks of LIPUS-stimulation [83]. These results imply that LIPUSenhances osteoclastogenesis throughout the entire time-course ofbone regeneration (Fig. 5, ph. 2,3,4), peaking on week 3, which cor-responds to woven bone resorption and lamellar bone formationin mice.
6.1.10. Immune responseImmune cells assure inaccessibility of any foreign particles in
the vulnerable cellular environment of the fracture, and, thus, per-form a prominent function in bone healing. The enhanced produc-tion of monocyte chemoattractant protein-1 MCP-1 (CCL2) andmacrophage-inflammatory protein-1 MIP-1b (CCL4) message wereobserved during LIPUS treatment of differentiating murine pre-osteoblasts [83]. The increase persisted from week 1 until week4, peaking on week 3. CCL2 and CCL4 are inflammatory cytokinesrecruiting monocytes, T-lymphocytes, macrophages and other im-mune cells, which potentially can maintain proper fracture healing[155] (Fig. 5 ph. 2,3,4).
6.2. Mechanotransduction Signaling Pathways Associated with LIPUS
Integrins, evolutionary conserved mechanoreceptors, are ex-pressed by various cell types and convert mechanical signal intobiochemical response [156]. Integrins have been proposed as keyplayers in the transduction of the ultrasound signal [15]. However,the type of activated integrins and their roles in the response toultrasound stimulation vary with cells types and origin as describedbelow.
6.2.1. Transmembrane mechanoreceptorsIncreased surface expression of a2, a5, b1 and b3 integrins and
clustering of b1 and b3 integrins have been shown to be upregu-lated within 24 h after 20-min treatment with LIPUS in rat primaryosteoblasts [114]. In the same cell type, but using continuous ultra-sound exposure, enhanced expression of a2, a5 and b1 integrinshas also been reported [131]. After ultrasound-exposure, geneexpression of a2, a5 and b1 integrins was also significantly up-reg-ulated in mouse osteoblasts isolated from long bones, whereasonly expression of a5 was enhanced in mouse mandibular and cal-varia-derived osteoblasts stimulated with LIPUS [157].
Inhibiting b1 integrin by blocking antibody or RGD-peptide inhuman primary skin fibroblasts, led to restoring basal levels ofDNA synthesis, which had been up-regulated in response to ultra-sound before [95].
Cooperative functioning of integrins a2b1 and syndecan-4receptors defines a successful outcome in wound healing [158].In mouse embryonic fibroblasts (MEF) ablation of the syndecan-4receptor (syndecan-4�/�) led to disruption of focal adhesion forma-tion when stimulated with the soluble syndecan-4 ligand [159].
However, when the mutant-cells were treated with LIPUS, cellsformed focal adhesions and activation of intracellular signalingpathways occurred. LIPUS elevated transient expression of GTP-Rac1 protein, signaling downstream from syndecan-4 receptor,and enhanced formation of focal adhesions in syndecan-4�/�
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mouse embryonic fibroblasts. Interestingly, PKCa protein kinase,mediating Rac1 activity in the presence of syndecan-4, is not in-volved in ultrasound-induced signaling events, and Mahoneyet al. [159] suggested that LIPUS acts downstream from these pro-teins or transmits its signal through unknown signaling pathway.The ability of LIPUS to substitute for syndecan-4 receptor and to in-duce focal adhesion formation through Rac1 activation has beenconfirmed by another study [160].
6.2.2. Pathways associated with PGE2 and NO signaling messengersCOX-2 expression, important for PGE2 production was found to
be regulated by signaling of focal adhesion kinase (FAK) MAPK ki-nase Erk1/2, PI3K and Akt kinases in response to ultrasound treat-ment in murine MC3T3-E1 pre-osteoblasts [114]. These signalingpathways were antagonized when the cells were treated with inte-grin inhibitors, and no enhanced phosphorylation of the proteinswas observed after the ultrasound-exposure, showing that inte-grins serve as an important link for converting mechanical signalinto intracellular signaling.
Signaling pathways involved in regulation of iNOS expression inmurine MC3T3-E1 pre-osteoblasts were also investigated. iNOS isanother important enzyme in bone metabolism. It was found thatultrasound induces iNOS expression via canonical NF-kB pathwayin the cells, which is preceded by activation of Ras, Raf-1, MEK,Erk and IKKa/b kinases [161].
6.2.3. Osteogenesis-associated pathwaysHuman periodontal ligament cells (HPDLC), which are similar to
mesenchymal stem cells, can undergo osteogenic differentiation[162]. Ren et al. [152] have reported that p38 MAPK kinase is crucialfor LIPUS-induced enhanced-differentiation of HPDLC cells. Treat-ment of cells with the p38 inhibitor significantly reduced ALP activ-ity, OC concentration and matrix mineralization in response toLIPUS, compared to the control group, where no inhibitor was added.
Naruse et al. [116] have demonstrated, using set of inhibitors,that enhanced differentiation of ST2 murine bone marrow-derivedcells, treated with LIPUS, could be linked to the PI3K and p38 ki-nases pathways, but not Erk1/2 MAPK kinase signaling. Ikedaet al. [163] has shown that the murine pluripotent mesenchymalcell line C2C12 can be committed to the osteoblastic or chondrob-lastic lineage when they are treated by LIPUS, through activation ofp38 and Erk1/2 MAPK kinase pathways. The discrepency betweenthese two studies about the activation of Erk1/2 signaling in re-sponse to LIPUS could be due to different cell types or to the inhib-itors used in the study of the Naruse et al. [116], which could haveabrogated expression of osteogenic markers. In the study of Ikedaet al. [163], direct effect on phosphorylation of the proteins wasexamined. The differences could be also attributed to the differ-ences in the US-set-up parameters, which are discussed in moredetail in the chapter 8.
6.2.4. Chondrogenesis-associated pathwaysThe MAPK/ERK pathway has also been implicated in a very re-
cent study [64] of bovine chondrocytes showing that blockingERK phosphorylation abrogate the low-intensity ultrasound-in-duced expression of early response genes c-Fos, c-Jun and c-Myc.Further transcriptional induction of these genes is frequency-dependent with highest c-series gene expression obtained at5 MHz as compared to 2 and 8 MHz.
Low intensity ultrasound in continuous mode caused more in-tense phosphorylation of FAK, Src, p130Cas, CrkII and Erk1/2 in pri-mary human chondrocyte culture, suggesting that this pathway isinvolved in US-induced mechanotransduction mechanism [81]. In-creased proliferation rate of primary pig articular chondrocytes inresponse to LIPUS has been linked to the integrin/PI3K/Akt path-way [82].
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6.3. Ultrasound-mediated modulation of the extra-cellularenvironment
Along with the direct effect of ultrasound, sensitizing mechano-sensitive receptors and channels of the cell, and the indirect effectof acoustic streaming-governed-shear stress on the cell surface,there is another effect, significance of which is often underappreci-ated. Acoustic streaming, giving rise to a unidirectional bulk fluidmovement, can improve the circulation of molecules within theextracellular matrix in the culture well, or trigger fluid flowin vivo, and thereby increase the delivery of cytokines secretedby other cell-participants or other essential nutrients, and removecellular waste products [164]. The accessibility of the crucial fac-tors to the compromised cells supports their viability and main-tains the indispensable microenvironment in the healing fracturethrough the regulation of pH, oxygenation etc. which may be en-hanced by the ultrasound treatment.
A mechanism of improved oxygen and nutrient transport in re-sponse to ultrasound has been suggested by Pitt and Ross [165],who observed the effect of acoustic streaming on different strainsof bacteria. Thicker biofilms were formed in response to low inten-sity low frequency ultrasound exposure (100-ms bursts at 2 HzPRF, 70 kHz ultrasound frequency, I = 2 W/cm2, type of intensitynot was reported). The same group had shown previously that com-bining 2 h of continuous ultrasound with antibiotic Gentamicincould eliminate up to 99% of bacteria, whereas treatment with theantibiotic alone, achieved only 82% [166]. Biofilms, formed duringbacteria population, represent a strong barrier against penetrationby oxygen, nutrients and more importantly, against penetration byantibiotics [167,168]. Lack of oxygen and nutrients slows downthe bacterial metabolism in the biofilm, decreasing their susceptibil-ity to antibiotics [169]. It is believed that improved transport of mol-ecules due to acoustic streaming, not only delivers antibiotics to thebacteria, but also paves their way through the film by ‘‘reactivating’’the bacteria and increasing the effectiveness of the drug [166]. Thisfinding is critical for patients with open fractures and/or receivingbone transplants or replacements, which could be at risk of bacterialinfections and biofilm formation. Therefore, complementary treat-ment of ultrasound with antibiotic therapy appears to be a potentialway of improving clinical outcomes for these patients.
Radial diffusion of Trypan blue dye was shown by Park et al.[170] using 22 kHz frequency, 30 mW/cm2 intensity and up to10 min indirect low intensity ultrasound stimulation. This groupreported that proliferation and uptake of glucose by adipose orga-noids was significantly increased, while TNF-a expression wasmarkedly decreased in response to ultrasound, indicating an in-creased metabolic activity. These effects could be explained byan improved supply of oxygen and transfer of nutrients to theorganoids, in situation when cells can have limited access to thediffusing molecules.
The assumption that acoustic streaming could be responsible foran increase mass transport and could trigger the aforementionedeffects is supported by other experimental evidences. Similarly tothe ultrasound-induced acoustic streaming effect, bulk fluid flowcan also achieved by using perfusion bioreactors. Static 3D culturesusually fail to reproduce the tissue-like morphology and function,because of the poor mass transfer to the cells in the center of thescaffolds and dynamic culture serve as a good alternative to that[171–173]. Oxygen concentration can directly affect cell viability.Moreover it has been shown that oxygen level can influence osteo-genic differentiation. Volkmer et al. [174] showed that MC3T3-E1cells grown for 5 days in 3D static culture have died in the centerof the scaffold, where oxygen level was about 0%, while survivingon periphery. When the same cells were grown in dynamiccondition, the cells death in the center was prevented, and oxygengradient was lowered. Furthermore, hMSCs, grown in hypoxic
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conditions, experience down-regulated-expression of osteogenicphenotype genes like Runx2, OC and Col-1 [171]. Improved masstransfer was also confirmed by Pisanti et al. [173], who showed thatmesenchymal cells grown in dynamic 3D culture exhibited a higherproliferation rate, and this seemed to depend on pore size, with big-ger pore size promoting higher rates. The authors hypothesized thatlarger pores improved the transport of nutrients and therefore in-creased the proliferation rate. The same authors found improvedosteogenic differentiation of cells under the dynamic conditions,which was evaluated by BMP-2 and ALP expression.
It should be noted that it is not always obvious to attribute theimproved differentiation of the cells to the increased mass trans-port or to the shear stress agitation of the cellular membrane. Todifferentiate these two contributors, Li et al. [172] manipulatedthe generated shear stress in a 3D-perfused hMSC culture bychanging the fluid viscosity at fixed flow rate or by changing theflow rate at a constant fluid shear stress. The study showed thatby increasing shear stress, ALP activity was significantly increasedon day 28 of culture at 3 ml/min flow rate. Increasing flow ratefrom 3 ml/min to 6 ml/min at the same shear stress also signifi-cantly increased ALP activity. Osteopontin secretion was enhancedin response to each of the two parameters, implying that bothmechanisms regulate osteogenic differentiation of hMSCs.
Since the essence of acoustic streaming is movement of fluid, itis most likely that its effect is most pronounced during the earlierhealing stage in vivo, i.e. during hematoma and soft callus forma-tion. The impaired vascularization at the fracture site cannot long-er fulfill the needs of the compromised cells. Similarly to aperfusion system, the ultrasound-generated acoustic fluid flow ishypothesized to provide the cells with oxygen and nutrients, res-cuing the viability and supporting the phenotype of the cells.Therefore, acoustic streaming may be an indirect, but yet promi-nent effector of enhanced bone fracture healing. The developmentof acoustic streaming is facilitated when the ultrasound propagatesin one direction. While this condition may usually be fulfilled forin-vivo applications, in-vitro systems, in which the sound is re-flected several times between the interfaces (Fig. 3a and b), mayprevent the development of an unidirectional fluid flow. Therefore,the aforementioned effects induced by acoustic streaming may notoccur in vitro in some cell culture stimulation systems.
7. Other forms of ultrasound treatments improving boneregeneration
LIPUS relies on a direct stimulation of the cells implicated withthe bone healing processes. We will review in this section otheruse of LIPUS synergistically combined with hormones or growthfactors, but also other forms of ultrasound that has been proposedfor the purpose of stimulating bone repair.
7.1. Synergistic effect of LIPUS combined with essential participants inbone and cartilage regeneration
Due to accelerated bone healing ability of LIPUS, there has beenincreased interest in combining the treatment with application ofother essential participants, known to have bone-healing efficacy.
Combined treatment of LIPUS and a calcium-regulating hor-mone, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), implicated withimproved fracture healing, has shown a synergistic effect on theexpression of PDGF on day 5 in co-culture of human umbilical veinendothelial cells (HUVEC) and human osteoblasts SaOS-2 [110]. Inthe presence of either LIPUS or 1,25-(OH)2D3, the production ofPDGF, which is an important factor for the regulation of MSCsand osteoblasts in the bone fracture, has been demonstrated.
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However, the highest effect was achieved when the two treat-ments were combined.
A significant improvement of chondrogenic differentiation hasbeen reported in response to treatment of LIPUS with TGF-b3 in a pel-let culture of hMSCs [175]. The hMSCs treated with both LIPUS andTGF-b3 exhibited more intense Alcian blue staining and about 3-foldincrease in aggrecan production, compared to the pellets solely trea-ted with TGF-b3. Similar results were reported in another study,where cells were grown in monolayer and TGF-b1 was used insteadof TGF-b3 [176]. Moreover, elevated Sox9 (a gene encoding for a tran-scriptional factor, prominent for chondrocyte differentiation),aggrecan, and Col-2 expression were observed in response to thecombined treatment. Intriguingly, in the first weeks of LIPUS-typestimulation of MSCs embedded in PGA scaffolds implanted in theback of nude mice, it has been observed that LIPUS up-regulatedchondrogenic differentiation of rabbit MSCs even in the absence ofTGF-b, before a dominant osteogenic area could be observed in thelater phase of the study (6 weeks post-implantation) [2,177]. Thesedata suggest that LIPUS preconditioning in vitro could be effective toup-regulate chondrogenic differentiation of MSCs implanted in scaf-folds in vivo for cartilage tissue engineering applications.
BMP-2 and BMP-7 are the morphogenes approved by the FDA(Food and Drug Administation) for the treatment of bone fractures.Despite their promising bone healing ability, numerous side effectsare associated with these cytokines [164]. Given the fact that BMPsare multifunctional players found throughout various tissues, thedose administration for a fractured bone should be stringently con-trolled. Due to growing knowledge of synergistic effect of LIPUScombined with various macromolecule treatments, simultaneousapplication of LIPUS and the BMPs appears to be an appealing toolin dose-controlled mediation of fracture healing.
In a rat ectopic implant in-vivo model, BMP-2, loaded on bovineCol-1 scaffolds, and simultaneous LIPUS application induced moreintensive bone formation after four weeks of stimulation, com-pared to rats treated with BMP-2 only [178]. Interestingly, so farthe synergistic combined effect of LIPUS and BMP-2 has not beenconfirmed in vitro. Stimulation of hMSCs combined LIPUS andBMP-2 treatment did not affect gene expression of Runx2, Col-1,Col-2 and ALP expression on day 3 [176]. Similarly, there was nopronounced influence of the treatments on Runx2 and ALP geneexpression, but a moderate up-regulation of Col-1 and osteopontinon day 3 and 5 in rat MSCs [135]. Thus, the mechanism of the syn-ergistic action of BMP-2 and LIPUS observed in vivo requires fur-ther in-vitro investigation. One of the possible signalingmechanisms may come from study of mechanical cyclic loadingof human fetal osteoblasts (hFOB), which reported a synergistic ef-fect with treatment of BMP-2 through phosphorylation ofSmad1,5,8, followed by nuclear translocation of the proteins andregulation of genes involved in bone morphogenesis [179].
In contrast to the aforementioned in-vitro experiments, com-bined exposure to LIPUS and BMP-7 has been shown to influencethe osteogenic differentiation of human hematoma-derived pro-genitor cells [178]. As a result of the treatment, increased expres-sion of ALP, Runx2 and OPN was reported on day 21, OC on day14 and 21 as well as elevated ALP activity on day 14 comparingto the cultures treated exclusively with BMP-7.
Not only vitamins or growth factors have been investigated tohave a potential effect on accelerated fracture healing withsimultaneous LIPUS exposure, but also MSCs, inevitable cellsin bone regeneration, have shown to have an improved healingeffect when combined with US in a rat model [180]. Rat GFP-labeled MSCs were exogenously injected and migrated to thefracture site, independent of the LIPUS treatment. However, inthe group where MSCs were combined with ultrasound, thefracture healed faster, which was assessed by quantification ofcallus width and area and value of bone volume to tissue ratio.
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Treatment with MSCs regardless of LIPUS presence resulted infaster bone remodeling.
LIPUS have also been combined to gene therapy. In a mice mod-el of ectopic bone formation in the muscle, gene transfer of BMP-4by electroporation and subsequent application of LIPUS resulted inan accelerated maturation of the ectopic bone formation [181].
7.2. Acoustic shock waves
Acoustic shock waves have been studied for their potential topromote beneficial therapeutic effects on different bone-relatedclinical complications. Acoustic shock waves treatments are verydifferent from LIPUS-type ones, due to the very different natureof the stimulation applied in tissues [29]. A shock wave is ashort-duration (<10 ls) acoustic pressure wave consisting of acompressive phase (peak pressure: 30–100 MPa) followed by atensile phase (negative pressure). When propagating into tissues,it will be associated with the generation of very high compressive,tensile and shear stresses, as well as generation and collapse ofbubbles (inertial cavitation). Shock waves can be generated by dif-ferent types of sources: electrohydraulic, i.e. a spark source whichgenerates a shock wave that is focused by an ellipsoidal reflector,electromagnetic, i.e. using an electrical coil in close proximity toa metal plate as an acoustic source, or piezoelectric, i.e. using alarge focused transducer. In shock wave lithotripsy, acoustic en-ergy is focused to a relatively small zone surrounding the focalpoint of the lithotripter, an elongated, elliptical ‘‘cigar-shaped’’ vol-ume of typically a few tenths of mm in length and a few mm inwidth. A treatment, in lithotripsy to induce kidney stones commi-nution, and in application of shock waves to bones, will consist ofthe application of 500–2000 consecutive shock waves, at repetitionfrequencies of typically 1 Hz. Historically, the application of shockwaves to medicine was lithotripsy where stones that form in thekidney, bladder, ureter, or gallbladder are fragmented into passablesize by shock waves. Shock wave lithotripsy has become a standardprocedure in urology. Applications in the bone context are more re-cent and shock waves have been used for the treatment of non-un-ions and pseudoarthrosis [182], the loosening of bone cementduring revision arthroplasty [183], bone regeneration in hip necro-sis [184], healing at the bone–tendon junction [185], and enhance-ment of bone callus formation during bone lengthening [186]. Todate, they are currently two FDA-approved indications for theuse of shock waves in orthopedics, treatment of plantar fasciitisand lateral epicondylitis [187]. In the treatment of bones fracturesand femoral head osteonecrosis, shock waves have been reportedto promote bone remodeling but also angiogenesis [188,189] andbeing able to restore the healing process in cases of non-union[190]. Mechanisms of action are possibly related to the inductionof micro-fractures in bony tissues due to the very high stresses in-duced by the propagation of shock waves [190,191], that in turncould trigger the initiation of remodeling cycles; the stimulationof neovascularization [192,189], possibly associated with aninflammatory response following induced trauma in soft tissuesdue to shock-waves induced inertial cavitation phenomena[193]; together with direct effects on cells proliferation, membranepolarization, expression of bone morphogenic proteins and activa-tion of cascades of mechanotransduction pathways [188,194–197].
7.3. Ultrasound and tissue engineering
Ultrasound has also shown potential in bone tissue engineeringstrategies. Engineering tissue regeneration relies on the delivery ofbiologics, i.e. cells, signaling molecules, genetic materials, viadegradable and non-degradable scaffolds [198]. The process of tis-sue engineering is therefore often described as a ‘‘triad’’, includingscaffold, cells and microenvironment (signaling molecules and
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physical environment). Ultrasound has played, or has a potential toplay a role in all these aspects of tissues engineering processes.
For the biologics: ultrasound can be used to promote cell prolif-eration or to pre-condition cells to orient their differentiation dur-ing culture, as reviewed earlier with LIPUS, but also to transfectcells. Ultrasound associated with microbubbles, such as acousticcontrast agents, a technique aiming at transiently altering the per-meability of cell membranes – so called sonoporation, is a powerfulway to induce internalization of genetic materials [31]. This meth-od has been applied to induce ectopic bone formation by sonopo-ration of naked DNA encoding for an osteogenic gene (rhBMP-9)into mice thigh muscles [3]. In vitro, transfection with ultrasoundand microbubbles has also been used to genetically modify MSCsbefore their implantation with siRNA to knock down PTEN mRNAexpression and activation of Akt, a mediator of a survival signalingpathway [199].
Furthermore, ultrasound can be used for the monitoring and thecontrol scaffold degradation rate, for the manufacturing of scaf-folds, for the characterization of scaffold properties and for theirquality control, and for improving scaffold integration [7–10].
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7.4. Ultrasound-triggered delivery of growth factors
Ultrasound can be used to controlled delivery of growth factorsor the gene expression of engineered cells, but also to modulate thephysical environment by heat deposition or mechanical stimula-tion, in order to promote regeneration. Delivery of bioactive mole-cules as instructive cues to engineered tissues can also benefit fromthe specificity of ultrasound-mediated delivery techniques. Micro-bubbles have been combined with growths factors for a spatio-temporal control of their release [4]. We recently developed adelivery system composed of fibrin hydrogels doped with growthfactor-loaded double emulsion for applications in tissue regenera-tion and on-demand release of growths factors [200].
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8. Discussion of conflicting published results
Numerous in-vivo and in-vitro studies have demonstrated thepositive role that ultrasound can play in the enhancement of frac-ture healing or the reactivation of a failed healing process. Thereare several modalities for the use of ultrasound in this context toinduce a direct physical effect (LIPUS, shock waves), to deliver bio-active molecules such as growth factors, or to transfect cells withosteogenic plasmids. We have focused in this review mainly onthe LIPUS-type stimulation of fracture healing, as it is the mostinvestigated and widespread application to date.
The biological response to LIPUS is complex, as different bio-ef-fects can be induced either directly or indirectly by the acousticwaves and various cell types respond to these stimuli in a con-certed manner involving several pathways.
Among the difficulties to understand the mechanistic basis ofthe observed stimulatory effects are the technical limitations bywhich ultrasound has commonly been used in vivo and in cellculture stimulation systems. LIPUS has been introduced as asimple and wearable in-vivo device that supports and acceleratesthe healing of human bone fractures [201]. The dimension of thesound field produced by the flat circular transducer ensures easyhandling and deposition of the majority of the acoustic energy inthe fracture repair region. Driven by the encouraging clinicaloutcomes, the same or similar systems have been used to inves-tigate the mechanistic effects of LIPUS stimulation in vivo insmall animal models and in vitro in cell cultures. Although aplethora of effects have been observed so far, the reports are atleast in part contradictory and hinder a general acceptance ofthis method.
Please cite this article in press as: F. Padilla et al., Stimulation of bone repair withttp://dx.doi.org/10.1016/j.ultras.2014.01.004
In addition to the many biological factors, e.g. cell types, cultureconditions, co-stimulatory factors, the translation of the clinicalstimulation system to a small animal model or a cell culture sys-tem appears to be challenging due to the physical dimensions ofthe acoustic sound field and the various physical effects inducedby the propagating waves. Whereas for the typical application inhuman fractures the ultrasound waves are predominantly emittedin the fracture repair region, the same device irradiates the entirebone including surrounding soft tissues in small animals. Whereasin-vivo applications may prevent the development of standingwaves due to the irregular organ and tissue morphology resultingin divergence, scattering and attenuation of the emitted waves, theregular dimensions and interfaces perpendicular to the soundpropagation direction as well as the low attenuation in the culturemedium and culture dish materials lead to multiple reflections,standing waves and potential heating artifacts. Therefore, for a par-ticular ultrasound transducer that emits a well-defined acousticintensity pattern in an interface-free fluid, not only the acousticintensity but, also the induced physical mechanisms may beremarkably different between in-vivo and in-vitro conditions oreven between in-vitro experiments with different exposure geom-etries. In consequence, care must be taken, when comparing thefindings of different LIPUS studies.
There are several set-up parameters, which have been shown tohave controversial effects in in-vitro settings and should be takeninto account in order to achieve agreeable data in response tothe ultrasound treatment. The geometry, affecting transmission,reflection, as well as spatial and temporal distributions of pres-sures and intensities exerted on the cells during in-vitro experi-ments, are factors that can jeopardize the results of experiments.The physical characteristics, e.g. ultrasound frequency, intensityand duty cycle, and duration of application of the applied ultra-sound beam have indeed been reported to potentially affect theoutcome of the stimulation, both in vitro and in vivo.
For example, using similar frequency and temporal settings(1.5 MHz center frequency, 200 ls pulses, for 5–20 min daily), Tsaiet al. [202] found that while 0.5 W/cm2 (ISATA) significantly acceler-ated bone repair, 1.0 W/cm2 suppressed it. Similarly, Reher et al.[203] reported that while 0.1 W/cm2 stimulated collagen andnon-collagenous protein synthesis, 0.5–2 W/cm2 inhibited it (at3 MHz center frequency, 2 ms pulses for 5 min). Similar datashowed that increasing ISATA from 30 mW/cm2 to 150 mW/cm2
did not improve bone volume fraction or failure torque in a modelof closed femoral fractures in rats [204]. Frequency has also beenreported to play a role: Wang et al. [205] reported that 1.5 MHzcenter frequency, but not 0.5 MHz, could accelerate fracture repairwith greater stiffness on a rat model of closed femoral shaft frac-tures, all other parameters being constant (30 mW/cm2, 200 lspulses, 1 kHz PRF). The PRF might be a key player in the optimiza-tion of the LIPUS signal, as changing the PRF from 1 Hz to 1 kHz hasbeen shown to affect the biological response to LIPUS [49].
Discrepant effects on osteoclastogenesis have been reported inresponse to short-burst versus longer-burst or continuous modeof ultrasound stimulation. Maddi et al. [132], using continuouswave 45 kHz ultrasound, have shown that OPG gene expressionwas elevated 0, 12, 18 and 24 h ultrasound post-exposure in oste-oblasts, whereas RANKL level has not been changed in response tothe treatment. These results suggest that osteoclastogenesis isinhibited by continuous wave ultrasound treatment, which is inagreement with Yang et al. [131], who showed that 1-MHz ultra-sound in continuous wave mode reduced the number of tartrate-resistant acid phosphatase (TRAP) – positive osteoclasts culturedfrom rats femoral bone marrow. Sun et al. [115], using a 1-MHzpulsed wave mode, but at longer pulse length (500 ls), demon-strated that osteoblast and osteoclast numbers increased and de-creased, respectively, in the co-culture of both, when treated
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with ultrasound. These findings support the hypothesis that osteo-clasts might be inhibited by exposure in continuous mode or burstswith long duty cycles. In contrast to these findings are results byHayton et al. [134], who showed that RANKL expression in osteo-blasts doubled, while OPG expression halved after 8 h LIPUSpost-exposure, concluding that osteoclastogenesis is rather acti-vated by ultrasound. These results coincide with Bandow et al.[83], who reported that RANKL expression was up-regulated dur-ing 4 weeks and no changes in OPG expression were observed indifferentiating MC3T3-E1 pre-osteoblasts in response to the LIPUSexposure. Both studies used the common LIPUS parameters(200 ls bursts at 1.0 kHz PRF, 1.5 MHz, ISATA = 30 mW/cm2) withand without an absorbing chamber, which may imply that osteo-clastogenesis is enhanced by short-burst ultrasound application.
Opposite effect of ultrasound have also been reported on thedifferentiation of pre-osteoblastic mouse MC3T3-E1 cells byUnsworth et al. [140] and Bandow et al. [83] using near field andfar field ultrasound stimulation respectively. The formershowed that using 1.5 MHz ultrasound burst of 200 ls at 1 kHzand ISATA = 30 mW/cm2, mRNA expression, activity of ALP, and ma-trix mineralization was enhanced after 10 days of treatment. Onthe contrary, Bandow et al. [83], using the same set of acousticconditions, demonstrated that neither ALP, Col-1 and OC geneexpression nor matrix calcification was affected by the treatmentwithin 4 weeks, bringing the authors to the conclusion thatultrasound does not affect differentiation of pre-osteoblastic cells.This contradiction in the effect of ultrasound on osteogenic differ-entiation may be explained by the fact that the stimulation in Uns-worth’s study has been performed with the transducer directlycoupled to the well-plate bottom (Fig. 3b), whereas Bandow’sexposure was in the far field with an absorbing chamber prevent-ing standing wave effects (Fig. 3c). Since heating, intensity gradi-ents, standing waves, and a lack of acoustic streaming can beconsidered in the set-up of Unsworth et al., but not in that of Ban-dow et al., thee observed differences support the importance ofproper control of the boundary conditions.
Another example of controversial biological outcomes inresponse to near field versus far field ultrasound exposure is adiscrepancy in gene regulation in response to LIPUS, shown byWarden et al. [125] and Naruse et al. [119]. Similar sets of condi-tions were described in the studies, using 200 ls ultrasound burstpulsed at 1 kHz with intensity ISATA = 30 mW/cm2, frequencieswere 1.0 MHz and 1.5 MHz in the former and the latter study,respectively. As a result of Naruse et al. [119], an up-regulatedtransient expression of c-fos could be seen 20 min after the stimu-lation, as well as elevated amounts of IGF-1, OC and BSP messagehave been shown to occur soon after the treatment, sustainingwithin 24 h post-exposure. Increased transient expression of c-fos is in agreement with Warden et al. [125], however, there wasno expression of IGF-1 and BSP observed within 24 h post-expo-sure and OC level was up-regulated only 24 h after the stimulation.The difference in the obtained results could be attributed to the se-lected cell line in the study. Rat UMR-106 bone-forming cells, withosteoblast-like features were used by Warden et al. [125], andmouse ST2 bone-marrow-derived stromal cells, which are able todifferentiate into osteoblasts were used by Naruse et al. [119].These cells were not only derived from different species, but alsorepresent cells in different maturation state, as it has been notedby Warden et al. [125], cells site-specificity could also alter theoutcomes of LIPUS-stimulation. Thus, Watabe et al. showed thatprimary mouse osteoblasts derived from mandible differently ex-pressed osteogenic resorption and survival markers after LIPUSstimulation comparing to osteoblasts from calvaria and long bones[157]. However, the differences in LIPUS stimulation set-ups, asdiscussed above, could also potentially contribute to the observeddifferences.
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These examples demonstrate the necessity to design experi-mental setups, in which acoustic peak pressure levels, temporalvariation of intensity, resulting radiation pressure and acousticstreaming can be well controlled. More important than the docu-mentation of the acoustic output levels are type, amplitude andduration of the ‘dose’ experienced by the cells. Furthermore, it iscrucial to develop studies, which can differentiate contribution ofeach of the previously described US bioeffects. These approachesshould help achieving more reproducible outcomes of cell stimula-tion studies and contribute to the understanding of the biophysicalmechanisms of LIPUS action.
9. Future directions
Given the current state of the art, there are different directionsthat might now be addressed to improve ultrasound stimulation ofbone formation.
The first ones are technological. The examples described in theprevious sections have demonstrated the necessity to carefully de-sign experimental setups and to document the acoustic output lev-els and more importantly the type, amplitude and duration of thestimulation ‘dose’ experienced by the cells. These issues can be ad-dressed in vitro, where acoustic peak pressure levels, temporal var-iation of intensity, resulting radiation pressure and acousticstreaming can be well controlled, and where side effects that canpotentially influence the response like standing waves or heatingby the transducer can be avoided. Systems operating in the far fieldof unfocused transducers with an absorbing chamber behind thestimulated cell layer seem to be the most predictable designs sofar. Controlling the sound propagation path length in the culturemedium could allow precise control of the acoustic streaming inthe stimulated well. Due to the complexity of the mechanisms in-volved, it will now be crucial to conduct studies that can differen-tiate the contributions of each of the previously describedultrasound bioeffects.
Alternatively to the use of plane wave transducer, focusing theultrasound wave to area similar in sizes to the dimensions of afracture or osteotomy in small animal models can also be used,similar to ultrasound focused systems used to follow up. With suchsystems, entire acoustic energy can be deposited in the repair re-gion by the focused beam, which we though should allow a bettercontrol of the deposited dose. Such systems are anticipated to pro-vide a better convergence of in-vitro and in-vivo results.
A second direction of research is the combination of ultrasoundimaging and therapy for both stimulation and monitoring of the re-sponse. Different approaches have been proposed to monitor bonehealing in vivo using ultrasound like focused through-transmission[206] or axial transmission [20,21,207,208] techniques that havebeen reported to distinguish fibrous or cartilaginous tissues frommineralized ones.
A third direction that merits attention is the passage fromin vitro experiments to clinical use. The passage from in vitro, tosmall animal models and then to large, weight-bearing and func-tional models is not trivial. Among the most challenging issuesfor the comparability of in-vitro and in-vivo studies are the differ-ent boundary conditions. While in-vivo bone regeneration involvesa plethora of cells of different origin (residing osteocytes, stemcells, immune cells and macrophages), which have to orchestratethe synthesis of tissue under highly compromised conditions, i.e.diminished nutrition and waste transport, inflammatory tissue re-sponse, disrupted tissue architecture, in-vitro conditions are usu-ally optimized to obtain a maximum cell response with respectto proliferation, differentiation, and matrix production. Under suchconditions, stimulatory effects may be much less pronounced orthe response may be different compared to the in-vivo situation.
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One of the advantages of the ultrasound technology, at least the LI-PUS one, is the absence of reported side effects. We can thereforeexpect that obtaining reproducible in vitro results in more complexbiological systems could pave the way for new clinical trials. Tomimic relevant conditions in-vitro, future studies should aim atworking with more complex biological systems and at compromis-ing the supplies of nutrition and growth factors and to investigatethe synergistic effects of cell–cell interactions. This can be achievedusing co-cultures, organoids or 3D tissue engineering seeding sev-eral cell types in scaffolds. These type of cultures will also allow toexplore the effects of ultrasound on increase mass transport, whichcould benefit tissue grafts during the immediate post-implant per-iod, when blood supply to the implanted tissue is suboptimal as re-ported recently [170].
Bhandari and Schemitsch recently argued in editorial of the JOrthop Trauma [209] that innovation in the management of bonefractures was likely to come from a ‘biologic—a drug or device thatfurther enhances the healing potential of fractures treated with mod-ern day implant fixation’, that could come at a reasonable cost. Inthis context, ultrasound technologies can have a role to play, dueto its therapeutic potential, possibly combined with monitoring,that comes at low costs compared to other non-healing fracturemanagement’s strategies. Improvements will probably come froma combination of ultrasound with a ‘biologic’ components, e.g.growth factors, scaffolds, gene therapies, or drug delivery vehicles,the effects of which could being potentiated by ultrasound. Ultra-sound-based drug delivery approaches are currently intensivelyinvestigated and is likely that combination of this approaches withthe intrinsic stimulatory potential of ultrasound will open the wayfor new applications to the orthopedic field.
10. Conclusion
Ultrasound can enhance fracture healing or reactivate a failedhealing process. There are several options available for the use ofultrasound in this context, either to induce a direct physical effect(LIPUS, shock waves), to deliver bioactive molecules such asgrowth factors, or to transfect cells with osteogenic plasmids.
We have focused in this review mainly on the LIPUS-type stim-ulation of fracture healing, as it is the most widespread and stud-ied. The biological response to LIPUS is complex, as numerouscell types respond to this stimulus involving several pathways.Mechanotransduction pathways seem to be involved in cell re-sponses, as demonstrated by the role of MAPK and other kinasespathway, the role of gap-junctional cell-to-cell intercellular com-munication, up-regulation and clustering of integrins, involvementof the COX-2-PEG2 pathway and activation of ATI receptor. Themechanisms by which ultrasound can trigger these effects remainintriguing. To some extent, these responses can be compared to theresponses to well-controlled mechanical stimulus such as shearstresses. Possible mechanisms include direct mechanical effectslike acoustic radiation force, acoustic streaming, propagation ofsurface waves; and indirect effects like fluid-flow induced mixingand redistribution of nutrients, oxygen and signaling molecules.Temperature is usually ruled out but its possible influence cannotbe neglected for some in-vitro set-ups. Although in-vitro studies arenot appropriate to identify the full complexity of biological effects,they are of interest to study specific mechanisms of action.However, great care has to be given to the design of dedicatedexperimental set-ups, in which the different ultrasound contribu-tions can be controlled. This will enable the study of the variousinfluencing parameters and to relate the variations of these param-eters to the induced biological responses. Then, it should becomepossible to derive an ‘acoustic doses’ required for particularresponses and to transfer such in-vitro findings to in-vivoapplications.
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Acknowledgement
The authors thank Ruslan Puts for his help in the design and forthe drawing of Fig. 5.
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