Department of Systems Solutions, Fraunhofer-Institute for High-Speed Dynamics, Ernst-Mach-Institut, EMI, Eckerstrasse 4, 79104 Freiburg, Germany; Department of Chemistry, Faculty of Science, University of Basel, Klingelbergstrasse 80, CH-4056, Basel, SwitzerlandSwitzerland
Also available as PDF (figures and tables appear in the PDF).
In the clinical treatment of solid tumors, besides traditional surgery and chemotherapy, the use of High Intensity Focused Ultrasound (HIFU) has been established as a minimally non-invasive technique for tumor treatment, which is based on coagulative necrosis of cells, induced by conversion of mechanical energy into heat. Another, less developed technique for the destruction or damage of tumor cells, is based on the pure mechanical effects of strong shock waves on cells, which are generated by using laser ablation, thus avoiding the heat-related unwanted side-effect when using HIFU (cutaneous burns of healthy tissue). Despite the general therapeutic success of extracorporeal shock wave therapy in medicine, e.g. for disintegrating congrements, the mechanical effects of shock waves on the cytoskeleton of cells, on the transient permeability and rupture of cell membranes, or on tissue damage remain widely unknown. The mechanical behavior of bio-macromolecules however, is of particular importance on the cellular level as several basic and yet unanswered questions are raised: How are cell stresses and energy transmitted through cells and in what way are the forces and interactions that determine the stability of cell plasma membranes affected by a shock wave and give rise to cell deformation, structural damage or rupture of the membrane with subsequent apoptosis? Here, we intend to review research on the shock wave destruction of tumor cells and discuss the use of laserablation as a new potential technique for tumor treatment. We also discuss here recent progress in computational modeling strategies and techniques for understanding the basic physical mechanisms that occur in the interaction of shock waves with cellular structures and show how computer modeling and numerical simulation can contribute to a fundamental understanding in this emerging multidisciplinary field, where physics, chemistry, biology and medicine meet.
Keywords: Shock waves; Molecular dynamics; Multiscale Modeling; Computer simulations; Cancer cells; Ultrasound; Laser ablation; Drug delivery
and under-express tumor suppressor genes (TMGs). Hence, the cells go through the cell cycle
these processes either by cytostatic (suppressing entry to or progression through the cell cycle) or cytotoxic (inducing apoptosis, programmed cell death) mechanisms. For example, chemotherapy agents such as doxorubicin are considered to be cytotoxic.2 Therapies that target hormone-addicted cells (e.g., tamoxifen in estrogen-driven breast cancer) are considered to be cytostatic.3
Studies of cells’ passive viscoelasticity-based biomechanics have already led to deep insight into a cell since its mechanical properties are directly related to its function by the cytoskeleton composition and architecture.4 Intracellular pathologic changes influence cytoskeleton structure and function, which makes a cell’s mechanical signature a highly sensitive marker of its health.5 In malignant cells the cytoskeleton devolves from an ordered and rigid structure to an irregular and compliant state, including a reduction of the cytoskeleton polymers and accessory proteins and a restructuring of the network. The cell changes from a mature state to a replicating, motile and cancerous form. From a more general viewpoint, cytoskeleton strength and organization increase as a cell becomes more differentiated. Thus, stem cells and malignant cells, as undifferentiated cells, are expected to be softer than mature and fully differentiated cells. Consequentially, measuring a cell’s rigidity provides information about its state and composition and may be viewed as a cell marker.5 In recent years, genetic, immunocytochemical and biochemical studies have identified many different players involved in the regulation of the cytoskeleton. Physical studies concentrating on the mechanical properties of cells or in vitro cytoskeleton model systems have been helpful in elucidating the complex synergies and redundancies of generic physical and specific biological mechanisms in establishing the overall mechanical performance of biological tissue. Unexpectedly, such studies have revealed a remarkable universality in the viscoelastic response functions of reconstituted networks and of whole cells of different types over a wide range of timescales, which is reminiscent of glassy 7,8 materials.7,8 At the same time, the overall cell stiffness has been demonstrated to be very sensitive to cytoskeleton dysfunction, a connection that lends itself to an extremely efficient and reliable automated detection (and may possibly also be the cause) of some diseases.5 To gain a detailed understanding of how these highly universal and specific mechanical properties of cells originate from the molecular structure of the cytoskeleton, it is essential to probe cell mechanics on multiple scales.
Traditionally, the only cure for solid tumors has been surgery, i.e. the resection of solid tumor tissue.9 However, open surgery is always associated with a high risk for the patient, suppression of a patient’s immune system and a significant mortality rate. Minimally invasive techniques as an alternative to open surgery, which became available during the 1990’s for localized tumor treatment, such as radiofrequency ablation,10 cryoablation,11 direct laser ablation,12 or high-intensity focused ultrasound
(HIFU), are methods which use a range of energies for direct in situ tumor destruction through energy absorption and killing by heating or evaporation. Besides direct conversion of mechanical energy into heat, biological material may be damaged by the effects of mechanical stress caused by shock waves.13 An efficient way to generate shock waves in biological systems became available to biomedical research by the use of high-power lasers shortly after the invention of the Q-switched pulsed laser in the 1960s.14
The theoretical foundations of shock wave physics originated th th from 17th century studies of classical acoustics and 18th century aeroballistics. With the advent of high-speed photography in late th 19th century and further progress in experimental visualization th techniques in 20th century, the study of shock waves in different states of matter has gradually emerged from a very small and unnoticed branch of physics to a major complex and interdisci- 6 15,16 plinary science.
Shock waves are in essence discontinuous, rapid mechanical phenomena that have been observed and studied in the laboratory and in nature, in microscopic as well as in macroscopic 17,18 19-21 dimensions and in all states of matter: gaseous, liquid, 22-24 25,26 27-29 solid, plasma, and even in Bose-Einstein condensates. Terrestrial examples30 of naturally occurring discontinuities are high-energy events such as meteorite impacts, thunder, volcanic explosions, sea-and earthquakes or tsunamis, while in outer space31 they encompass plasma shock waves induced by solar wind, supernovae explosions, implosions of white dwarfs, comet and asteroid impacts, and stellar or galactic jets. This variety of phenomena and in particular the possible applications of shock waves in different areas such as physics, chemistry, biology, medicine and even engineering generally renders the scientific study of shock waves a rather fascinating and interdisciplinary subject.
We focus here on the physical proper definition of shock waves which–in medical literature–is not always used correctly and many studies fail to show that they were really dealing with shock waves and not merely with high-energy sound waves. In the natural sciences, a shock wave describes a mechanical wave characterized by a surface or sheet of discontinuity in which, within a narrow region, thermodynamic quantities such as pressure p, density ρ, particle velocity v or temperature T change abruptly.17 While a theory of shock waves with an infinitesimal jump condition can be developed mathematically in the framework of approximate continuum models of condensed matter, i.e. based on the hydrodynamic equations of ideal fluids or gases, in physical context, shock wave theory is somewhat limited, because the width of a shock wave is always of finite size and 10 11 because of the actually discrete atomic nature of condensed matter. It turns out that shock waves are essentially small regions
of a system, where non-adiabatic, i.e. irreversible energy dissipation occurs. The notion of a shock wave as an infinitely thin sheet of discontinuity is a mathematical idealization. The width of the shock wave i.e. the size of the dissipative region establishes itself according to the conservation laws of continuum theory. In very strong, high-amplitude shock waves, their width becomes so small that they are practically indistinguishable from the mathematical idealization of an infinitesimally small perturbation with jump conditions for thermodynamic variables.
In solids, physical shock waves are mechanical waves of finite amplitudes and arise when condensed matter is subjected to a rapid compression. Shock waves can be defined by several major distinctive properties as follows:
• A pressure-dependent, supersonic velocity of propagation. • The formation of a steep wave front with a sudden change of thermodynamic quantities.
• Non-linear superposition properties (for reflection and interaction).
• A strong decrease of propagation velocity with increasing distance from the center of the origin for the case of nonplanar shock waves.
Sometimes another criterion for a shock wave is listed in shock wave literature which is the extremely short rise time of the pressure within tens of nanoseconds. However, this is not a defining criterion of shock waves, because no generally agreed upon definition exists of what “extremely short” really is supposed to mean – it is a term of rather subjective nature. A nanosecond rise time certainly applies for lithotripter shock waves which are used in medicine as a non-invasive technique to com- 32 33 minute calculi32 and for other therapeutic purposes33; however, in the geologic realm for example, meteorite impacts, explosive volcanic eruptions and earthquakes can provoke drastic and irreversible changes within seconds.
One characteristics that distinguishes a shock wave from an ordinary sound wave is that the initial disturbance in the medium that causes a shock wave is always traveling at a velocity greater than the phase velocity of sound in the medium (the electromagnetic analog of this is called Cherenkov radiation).34 Because a shock wave moves faster than the speed of sound, the medium ahead of the shock front cannot respond until the shock strikes, and so the shock wave falling upon the particles of matter initially at rest, is a supersonic phenomenon.
Since its invention in the early 1980s, Extracorporeal Shockwave Lithotripsy (ESWL) has evolved as a clinical standard, which is widely used as the only non-invasive surgical technique to eliminate e.g, kidney stones and other urinary calculi.33 Rassweiler et al35 describe the multiple mechanisms associated with stone disintegration by ESWL as well as current advances in in-
strumentation and clinical praxis. A shock wave is a mechanical wave that induces a transient pressure rise and propagates at a speed above the speed of sound of the corresponding medium. It is created by means of a piezoelectric, electro-hydraulic or electromagnetic transducer, called lithotripter, which generates focused high-amplitude pressure waves in a contact medium 35,36 outside the body. The patient is positioned such that the stone is brought into the focus of the pressure wave. When the acoustic energy deposited in the focal region is high enough, cavitation bubbles filled with vapor or gas form and collapse violently. This collapse results in formation of a shock wave that in turn disintegrates the stone. Apart from stone destruction, ESWL can cause tissue injury as unwanted adverse effects during treatment.37 The physical mechanisms related to the cavitation phenomenon that cause both stone destruction and tissue damage are very complex, not well understood and hardly experimentally controlla- 35,37-39 ble. The fact that ESWL can cause tissue damage, inspired experiments that applied lithotripter induced shockwaves to tumors in vivo. While several first studies in animal models did not show any impact of shock waves on tumor growth,36 some results seemed encouraging, for example a delayed tumor growth in mice40 and even complete remission of dorsal skin tumors in hamsters.41 However, up-to-date shock wave treatment has not been used in clinical traits, most likely because the cavitations phenomenon is very hard to control and the unspecific effects on ESWL on cells cause the need for live imaging techniques during treatment. Parallel to the in vivo experiments on tumors, a large number of in vitro studies on different cell lines in culture were performed as reviewed by Coleman and Saunders,36 and Delius.39
The application of several hundred shock waves created by commercial lithotripter systems causes cell death. Most striking for a possible shock wave based tumor therapy was a study on different normal and malignant cell lines, which showed no selective effect.38 When the energy deposited by the lithotripter is reduced below the lethal value, it is possible to permeabilize 42-46 the cells without killing them. In this way, molecules present in the surrounding medium can diffuse into the cells. This effect has possible applications for drug delivery and gene transfer.47 In more recent studies, adherently grown cells on transparent substrates were exposed to shock waves and analyzed with (fluorescence) microscopy. In direct vicinity of the cavitation bubble, the cells are completely destroyed and detached from the surface. Cells close to the bubble were permanently permeabilized and killed, whereas cells further away from the bubble 48,49 survived. This behavior could be connected with deformations of the cytoskeleton.50 Generally speaking, ESWL treatment of cells seems to be only poorly controllable because of the cavitation process and no selective effects on cells are observed. These disadvantages might be overcome by the technique of laser-induced shockwave generation discussed in the next section, which is based on purely mechanical destruction of cells, where no cavitation effects occur.
When irradiating an absorbing material with a pulsed laser, the optical energy deposited on the absorber is transformed into mechanical energy. A shock wave forms at the surface and then travels the absorber.51 The shock wave properties such as rise time, velocity of propagation or peak pressure depend on the absorbing material and the laser parameters. For one specific laser/absorber system, the peak stress of the shock wave can be tuned by varying the laser fluence that is equal to the total energy deposited per area of illumination. In this experimental configuration, well-defined, reproducible shockwaves can be generated without the side effect of heating or cavitation.55 Thus, the pure mechanical effect of the shockwave on the cells can be investigated. As reviewed by Yao et al,56 also Laser-Induced Shockwaves (LISW) are able to render the cell membrane permeable, giving rise to drug delivery and gene transfection. The application to whole tissue structures is also possible. For example, transdermal insulin delivery by LISW results in a reduction of the blood glucose level without causing any pain to the patient.57 Here, we focus on the direct lethal effect of LISW on cells. In the following, we review the literature concerning the impact of LISW on cells and present current progress and novel developed techniques that will help to further classify the properties of LISW.
In Table 1, we provide an overview of major studies
Lymphocytic mouse leukemia In vitro; cells in suspensions and immobilized in Dornier XL1 lithotripter; 58 cells L1210 gelatin; 125 to 500 pulses Human renal cell carcinoma RC-8, In vivo; series of 3-200 pulses with pulse grown in the mouse duration of 1.5 at 10Hz
Mouse breast sarcoma cells In vitro; 25-240 mJ pulse energy; up to 50 EMT-6 pulses Mouse breast sarcoma cells EMT-6 Human retinal pigment epithelium cells RPE
HeLa cells In vitro; 360 pulses Human bladder tumor cells HT- 1197
Human red blood cells RBC In vitro; 5 pulses
Mouse breast sarcoma cells EMT- 6 and human red blood cells RBC
Mouse tumor cells SW480 In vivo
Human endothelial cells HUVEC In vitro HeLa cells In vitro
Table 1. Overview of studies that involved the shock wave exposure of tumor cells. Unfortunately, as can be seen from this compilation of studies, no systematic investigation of biological shock wave effects in different types of cell lines with a very well-defined and reproducible method ever seems to have been conducted. Also the achieved maximum pressure levels of the shock waves and their rise times were not measured in all of the studies.
that investigated the biological effects of the interaction of shock waves (or strong pressure waves) with biological cells.
Figure 1 shows the basic features of the experimental 60-62,65,66,69 setups used in several studies reported in the literature. The laser beam is directed onto the bottom of a cell culture vessel, that consists of an absorbing material. Upon illumination with the laser, a shock wave is formed that travels into the vessel, and interacts with the cells. For one specific cell line, it turned out that the survival rates of cells exposed to LISW depend on 52-54 stress gradient σ=pmax/τR, where pmax is the peak pressure and is the rise time of the shock wave.60 However, the survival rates among different cell lines differ remarkably at constant physical parameters pmax and τR. For example, only 50% of transformed (immortalized) retinal pigment epithelium cells survive exposure to shock waves with pmax=74 MPa and τR=10 ns. However, 100% of normal retinal pigment epithelium cells survive this procedure.60 A shock wave with τR=10 ns and pmax=30 MPa kills 50% of mouse breast sarcoma cells, whereas human promelocytic leukemia cells survive this exposure.69
All of the above mentioned studies in Table 2 had major deficiencies in the techniques used to characterize the physical conditions in the vessel. Usually, the pressure profiles are measured with piezoelectric elements (Polyvinylidene fluoride, PVDF) either in form of needle hydrophones or piezoelectric films that are brought in contact to the surface of the culture
Brümmer et al 38.6 MPa Laser ablation (polyimide target) with a Candela LDFD/3 59 Reijke et al flash lamp pumped dye laser Laser ablation (polyimide target) with ArF/KrF excimer laser; Doukas et al 35-65 MPa Laser ablation (polyimide target) with ArF excimer laser; 61 In vitro Lee et al 30 MPa Laser ablation (polyimide target) with ARF/KrF excimer In vivo; 5-150 pulses laser; Douki et al 74 MPa, max. 240 mJ Siemens lithostar lithotripter; 63 Huber et al 69.5 MPa In vitro pulses; 500 Dornier HM-3 lithotripter Kohri Laser ablation (polyimide/polystyrene target) with Qswitched ArF excimer laser; Mulholland et al 60 MPa, max. 350 mJ Laser ablation (polyimide target) with ArF excimer laser In vitro and Q-switched ruby laser (polystyrene target) Lee et al 60 MPa Piezo-ceramic elements; 67 Kato et al 40 MPa Nd-YAG laser (copper target) 68 Sondén et al 23 MPa, max. 5.9 mJ ESWL, 28 MPa Ohl et al a) Basic elements of the experimental setups used to study the effects of laser induced shock waves on cells.36,49-53 (b)A
all previous studies reported in literature.
Polymer melts Entangled macromolecues
Crosslinked networks of Elastomers Dynamic moduli polymers Dilute solutions containing Solvent viscosity and intrinsic Models for solution and polymer 82 Polymer solutions polymer chains viscosity
Gels with polymers Yield stress, elasticity Network with crosslinks or particles yield), microrheology
Suspensions, emulsions, micellar Particles in fluids solutions
Table 2. Rheological properties of a few complex systems that can be used for upscaling in multiscale modeling of cellular soft matter.
vessel. In both cases, a transfer medium (either water or grease) serves as the acoustic contact to the piezoelectric element. However, shock waves are known to decay very rapidly (within micrometers) in liquids and tissue.70 Thus, the need for a fluid as contact medium may lead to wrong pressure measurements and it would be desirable to determine shock wave properties on a microscopic scale, rather than with a PVDF sensor of millimeter dimensions. An alternative to determining the pressure profiles with piezoelectric element is to use optical methods that measure the velocity profile of the vessel bottom while the shockwave passes. From this velocity profile the pressure profile in direct vicinity of the bottom plate can be calculated. This informationis particularly interesting when studying adherently grown cell cultures. Strand et al71 developed a cost efficient and compact high-speed velocimetry system that uses a heterodyne detection method.
of the vessel (usually a polymer) that contains the cells and their formed that moves forward into the vessel. When the absorbing
deGennes Dynamic moduli, via shear, Reputation theory, viscoelasticity, Doi and Edwards viscosity Maxwell model, Dumbbell model Bird Larson Gel-type models, yield stresses Winter and Chambon
Bird et al separately Flory (below 76 Percolation, fractal dimension deGennes Winter and Chambon Percolation, particle orientation, 86 Yield stress, viscosity, Einstein small-deformation theory, yield 87 diffusion Oldroyd stress
An optical fiber is used to transport Laser light to a probe that contains a lens. The light is then focused onto the moving surface that reflects or scatters light back into the probe from where it is brought to a detector. The frequency of the light is now Doppler-shifted because of the surface movement. At the detector, it is mixed with non-Doppler shifted light to generate a beat signal that contains the velocity information of the moving surface. In a recent breakthrough application, velocity profiles have been measured in this way and could also serve as input for simulations that computed the pressure fields within the whole vessel with high time resolution.72 In this way, the conditions that the cells are exposed to during the shock wave experiment can be characterized on the relevant length and time scales which are micrometers and nanoseconds. For beam diameters of a few millimeters, in the author’s laser lab, with an optimized experimental setup, peak pressures up to approximately 120 MPa were
obtained which is considerable beyond the pressure levels of all previous studies discussed in literature, see Figure 2.
Exploring the potential of shock waves for destroying or damaging cancer cells possibly opens a new or complementary road for tumor treatment, avoiding the disadvantages of currently established methods for tumor treatment based on US, and using only the mechanical destructive effects of a shock wave interacting with cells. By way of using the mechanical effects of shock waves rather than relying on heating of tissue, it seems possible to destroy or damage the cytoskeleton or the membrane, as shock waves carry much more energy in a much shorter time interval than US waves do. This might open new perspectives for cancer therapies in addition to existing treatments. The complexity occurring in the interaction of shock waves with soft, biological matter requires a combined approach using experi-
culture plates were used (positioned here on an optical table) and U87 glioblastoma cells were grown adherently on the bottom of the individual wells. The achieved pressure levels were measured directly in the wells by means of PVDF hydrophones with nanosecond time resolution. (b) By using black varnish as absorber material, it was possible to reach pressure levels in the cells beyond 100 MPa which is well above the pressure levels achieved in all previous studies reported in literature. For U87 glioblastoma, it was found that a biological destructive effect occurs above 80 MPa, which is a pressure level well above the ones achieved in all previous experimental studies reported in literature, cf. Table 1. One great advantage of the setup shown here, is its reproducibility in both, the pressure level measurement by using very well-defined single laser pulses per well and the level of destruction in the cells which was verified by cell counting and a long term MTA-analysis tive results (i.e. no destruction) in the untreated controls.
To model the mechanical properties of eukaryotic cells, a focus is set on the two salient features of cells that determine their mechanical behavior: the cytoskeleton (composed of a network of macromolecules) and the plasma membrane (composed of double lipid layers).
Considered from a physical perspective, living organisms such as eukaryotic cells or neoplasia are far more complex systems than typical engineering materials such as metals, ceramics, polymers or semiconductors, see Figure 4. They are dynamic and provide integrated functions that include metabolism, reproduction, growth, sensing, communication, control, and apoptosis (programmed cell death).
Different studies have established the connections between structure, mechanical responses and biological functions of different organs and tissues including, for example, the heart, lung, bone, cartilage, blood vessel, and skeletal and cardiac mus-
Optimized setup used in recent breakthrough experiments by Schmidt et al.72 (a) Ordinary multiwell cell
over several days, i.e. over several cell cycles with negacles.74 These studies have led to better diagnosis and treatment of orthopaedic, cardiovascular and respiratory diseases by providing a greater understanding of how the biological functions of the body are related to bio-solid and bio-fluid mechanics. To decipher the fundamental mechanisms of biological materials, however, more systematic studies of deformation, structural dynamics and mechanochemical transduction in living cells and biomolecules are needed. The mechanics of biological molecules, including proteins and nucleic acids, is crucial to understanding the connection between structure and function in cellular and tissue mechanics. Still in its infancy, this emerging field of bio-mechanics strives to understand the deformation and mechanics of macroscopic tissue structures based on the deformation and mechanics of bimolecular. A recent summary of current multi scale simulation approaches for bridging the scales from sub-cellular components to tissue is provided in a recent review by Steinhauser.73
Upscaling of atomistic and microscopic simulation approaches of the kind as displayed in Figure 5, is one way of trying to understand the basics of shock wave effects on soft matter systems such as cells and living tissue.75 There are a number of complex microscopic systems, which are relevant to the study of animal or human cells, and which form the basis of any physical, and mathematical-numerical modeling, since the systems we are interested in build a complex system made of polymer melts and solutions, suspensions, gels and micelle systems. The microstructure of these systems is very important for the elaboration of constitutive equations on a coarse-grained, phenomenological level.
In Table 2, we review and summarize some of the key rheological properties of a few of these systems. For example,
in a cell in the form of proteins and play a fundamental role for many cell functions and cell-cell interactions. The basic microstructure of a polymer network consists of chains intermingled with each other (entanglements) with a few weak reticulation points, as well as loops or dangling ends.76 Gel materials are interesting systems, because the cell cytoplasm may be regarded as a gel. Elastomers are slightly different and may be considered as viscoelastic solids, in particular, because they cannot flow at very low shear rates. This is mainly due to strong links (covalent sometimes) associating polymer chains thus creating a network which behaves elastically over a wide range of shear rates.
On the nanoscale, their microstructure looks something like a regular net. As the frequency is increased, they undergo a n glassy transition where moduli G' and G'' behave as ωn, where n is an exponent whose value is close to 0.6. Polymer solutions are solutions containing polymers in a solvent and do not exhibit entanglements in this regime. They may be considered to have two components, one being the solvent (which is viscous with constant viscosity η) and the other one being the polymer with viscoelastic properties. The field of suspensions is quite large, because it can describe particulate suspensions, but can also lead to fluid-fluid suspensions called emulsions, and all kinds of systems including deformable objects in a fluid. For example, blood is a mixture of white and red blood cells, platelets and other constituents included in the plasma.
Most of the biological cells are 1-100 microns in size, and they comprise many constituents, Figure 5. The cell is covered by a phospholipid bilayer membrane reinforced with protein molecules, and the interior of the cell includes a liquid phase (cytosol), a nucleus, the cytoskeleton consisting of networks of microtubules, act in and intermediate filaments, organelles of different sizes and shapes, and other proteins, Figure 5. The re-
network.79 Color code indicates forces in the bonds of the polymers with red: very large force just before bond failure, and green: force-free bonds.
particle amphiphilic lipid model (hydrophilic part of the lipid: red, hydrophobic part: yellow) where the lipids self-assemble to form bilayers. Subsequently, these equilibrated bilayers are exposed to strong shock waves.81 Simulation snapshot adopted from Schindler et al.80 sistance of single cells to elastic deformation, as quantified by an effective elastic modulus, ranges from 102 to 105 Pa, see Figure 6, which is orders of magnitude smaller than that of metals, ceramics and polymers. The deformability of cells is determined largely by the cytoskeleton, whose rigidity is influenced by the mechanical and chemical environments including cell-cell and cell-Extracellular Matrix (ECM) interactions.
If the elastic, visco-plastic properties of the basic constituents of cells and tissue are known, there is a variety of techniques to produce constitutive equations for a macroscopic description, such as ensemble averaging, mathematical ho- 90 91 mogenization,90 effective medium theory,91 or temerity models which are based on the idea to model deformable structures by using sticks and strings under tension.
(c) A simulation of the self-assembly of a three-
It was shown that this approach is particularly well adapted for the description of eukaryotic biological cells.98 However, the previously listed techniques are usually not able to account for specific interactions between cells, their individual, specific behavior or the active response of cells to external stimuli.
Another, more direct way of modeling shock wave effects in tissue and cells is to use macroscopic models based on continuum theory rather than upscaling basic properties. Continuum models, e.g. based on finite elements meshes neglect almost 88,89 all details of the discrete nature of the constituents of cells and tissue but allow for a macroscopic simulation of deformation upon exposure to shock waves. The quality of these type of sim- 92-97 ulations however, is very strongly depending on the employed
phenomenological material model and additional complexity is to be considered here, because of the local heterogeneity of tissue and a general lack of experimental measurements in vivo. Generally speaking, it is at least questionable, whether experimental in vitro studies of tissue behavior have real physiological relevance.
For many years, researchers have devoted their attention to the study of animal tissues, and important issues have been raised. Finding constitutive relations for such media is not simple, because tissues can behave as elastic, plastic, viscoelastic or viscoplastic materials. One of the most important conclusion is that relating the microstructure with its macroscopic nature is a fundamental problem which forms the basis of any continuum mechanics problem.83 The relevant sciences studying such aspects 83,99,100 101-104 are rheology and biomechanics.
These two fields are actually very close to each other when it comes to dealing with biomaterials, and defining their minor differences here is not my intention. One may say that generally one is interested in finding relationships between the applied forces and the relevant deformations or flows involved in problems dealing with living materials. Classical models (1D), which can be used and can depict the cytoplasm of a cell, are usually viscoelastic or viscoplastic ones. 3D-viscoelastic models can exhibit differential forms, or integral formulations (sometimes equivalent). Other models like viscoplastic ones can also be interesting because they allow us to deal with systems with cross-links, somehow close to gels; in particular, polymers and networks play a role inside the cytoplasm, Figure 5a and 5b. So, at a certain level, we may assume that the size of the system studied is large enough , where is the size of an element at the microscopic level, so that the system exhibits a macroscopic, averaged behavior and can be described using a constitutive relation as it is done in typical, empirical engineering approaches for material modeling.
Generally speaking, material models have to include the following material behavior (listed in increasing order of complexity):
1. Linear elasticity.
2. Hyperelasticity.
3. Hyperviscoelasticity.
Many computational approaches only include hyperelasticity. The general description from which such models start, is the Ogden hyperelastic material model105 which assumes the existence of a strain energy function in the form:
n p p p p µ α α α E = λ +λ +λ − 1 2 3 3 ∑p=1 ( ) αp αp where are the principal stretches, and are mateλα i p n rial constants, and μp denotes the shear modulus of the material
in the undeformed state. We note that the often used Mooney- 106,107 Rivlin material model, which assumes that can be written as the sum of two invariants, is contained in the above formula as a special case.
Therapeutic technologies such as focused US and shock wave treatment, have an extremely localized area of therapeutic effect and therefore have to be applied directly over specific location of anatomic/functional abnormality, precisely in relation to the current (i.e. intra-operative) patient’s anatomy. As surgical intervention tends to distort the pre-operative anatomy and often leads to misalignment between the actual position of pathology and its position determined from pre-operative images, an image-guided surgery requires intra-operative images and/or update of the pre-operative images to the current position of the brain internal structures. To achieve an accurate image update, deformations of the abdominal organs (e.g. the brain, the liver or the kidneys) must be taken into account. Since the late 1990s, significant research effort has been directed towards the predic- 108-115 tion of such deformations using biomechanical models. 116-119 Typically, in such models, the Finite Element (FE) method is employed to discretize and solve the related differential equations of continuum mechanics, see Figure 7. During imageguided surgery, only low-quality, sparse information about the current tissue position is available.120 This information can be used to warp high-quality pre-operative images, such that they correspond to the momentary situation, by computing the deformation field within the brain using a biomechanical constitutive model.
In a comparative study by Wittek et al114 it was shown that the actual choice of a specific material model hardly influences the calculated displacements of tumors. Between different models, the differences in displacement amounted to less than 0.2 mm. It seems that the organ displacement is dominated by its weak compressibility rather than the resulting stresses. Many different experimental setups and different theoretical descriptions are used to extract mechanical parameters from tissue measurements. We end this review by listing the results of various such studies in Table 3. One way of comparing these different results is to calculate Young’s modulus based on local linear elasticity.
As a clinical tool, within its limits of applications, HIFU is established as a therapeutic tool. As this technique becomes more widely available, it should be possible to coordinate the type of clinical trials that will be necessary to develop the evidence base for the efficacy of HIFU in its various applications, whether alone or in combination. Real-time imaging and treatment monitoring are the subjects of ongoing theoretical research, and the development of techniques such as three-dimensional ultrasound α and elastography which measures the change in stiffness of a tis p sue as it is ablated, are likely to enable improvement in clinical
Rabbit (in vitro) Indentation Deer (in vitro) Indentation, max 20 mm Linear/nonlinear/viscoelastic 25 Schwartz et al Porcine (in vivo) Aspiration Hyperelastic, viscoelastic 90 Nava et al Procine (in vivo) Indentation, max 500 µm Linear 10-15 Ottensmeyer Tissue scaffold (in vitro) AFM nanoindentation Porcine (in vivo) Indentation, max 8 mm
Table 3. Mechanical, experimental data on the liver of several studies that may be used as input and for validation of computer simulation approaches that mimic the mechanical behavior of biological tissue.
outcome and to bring about a reduction in treatment duration. In addition, focused ultrasound has been proposed as a vehicle for delivering targeted gene therapy through inducing cavitation of DNA-laden micro bubble contrast agents in the periphery of the zone of ablation, where temperature rises would be sublethal, but these remain secondary considerations to the direct ablative treatment intent at present.
The experimental observation that shock waves are able to destroy cells in vitro and to delay tumor growth in vivo motivates further studies of the responsible mechanisms. The author believes that characterization of shock wave impact on the cellular level is necessary to understand these processes more systematically. Laser-induced methods for shock wave generation have proven to be reproducible on the macroscopic level and to avoid lithotripter related side-effects such as heating and cavitation. Newly developed optical methods together with multiscale modeling approaches can give insights on the precise conditions that the cells are exposed to during shock wave impact. The knowledge of these conditions is a prerequisite to find and maybe control selective effects oncells. The diverse behavior shown by different cellular strains poses the need to find quantities that
the computed field of irreversible volumetric expansion in an exterior view. Meridional section of a anatomically correct finite element mesh of the kidney. The photograph illustrates how difficult it is to generally quantify the amount of tissue injury.
5.6 Yamada
11.6 Zhu et al 13 Tay et al
relate cellular properties with shock wave induced killing. Thus, in parallel to shockwave experiments on different cell lines, specific quantities have to be found that relate cellular properties with shockwave impact. Of course, mechanical properties related to the cytoskeleton serve as a first promising candidate and may by determined with combined cell-mechanical and fluorescent microscopic observations. In addition to the experiments on cells, another possible application of shock waves emerges from current biological findings that relate tumorous growth to mechanical properties of tissue. It has long been known that tumors are more stiff than healthy tissue. This fact is even used to detect mammary cancer, for example by palpation or elastography of rigid tissue structures. The mechanical properties of tissues are mostly influenced by the ECM, which mainly consists of collagen. These molecules are produced inside the cells and delivered to the ECM where they form polymeric structures that serve as scaffolds for the cells. When healthy tissue turns into a tumor, the polymeric structure of collagen fibrils is significantly altered, causing a stiffening of the ECM.81 Cells possess mechanoreceptors that detect physical forces and transform them into biochemical signals.82 Thus, cells are able to both sense and alter the mechanical properties of the ECM.83 Interestingly, the bio-
chemical signaling pathway of the mechano-sensing apparatus cross-talks with major pathways that control cell proliferation and it was shown that tissue stiffness promotes cancerous cell growth.84 In this way, ECM stiffening and tumor growth form a positive feedback loop that enhances the risk of tumor formation.82 On the other hand, these current findings may open new possibilities of cancer therapy basing on mechanical methods, i.e. based on the physics of cancer rather than or in addition to pharmaceutical treatments. One of these interesting possibilities of shock waves therapy based on mechanical effects alone, is its potential to be used for gene transfection and molecular drug delivery, which involves interesting physical chemistry research concerning the structure of molecules to be used as drug carriers.85
Within the biological sciences, cancer is highlighting the need for interdisciplinary research. There is a growing realization that cancer is a problem requiring input from a wider community of scientists, i.e. physicists, mathematicians and computer engineers. This is reflected in increasing interdisciplinary collaborations around the world, involving biologists, clinicians, physicists, mathematicians and computer scientists, which aim to give new insights into cancer and improvements in its treatment.
In a fashion quite analogous to studies of mechanics of engineering materials, the emerging field of cell and molecular mechanics of biological materials seeks to establish essential linkages between microscopic structure and macroscopic mechanical properties. New multiscale computational approaches have emerged in recent years, coupling e.g. atomistic scale simulations with the macroscopic domain as reviewed in Steinhauser76 and also new numerical approaches combining coarse-grained macromolecular models of bio-molecules with shock wave physics emerged.84 Although methods have been developed to measure cell responses during deformation, celladhesion, locomotion and mitosis, reliable experimental tools are currently unavailable for quantifying the distribution of mechanical forces between various subcellular structures as well as on individual proteins and nucleic acids inside a cell.
In summary, progress in experimental and computational biology and biomechanics during the past decade has provided unprecedented opportunities to probe the mechanical responses of cells, proteins and DNA molecules. How the forces and deformations associated with these basic structural units of life can be described by physical models, engineered by chemistry and implemented in computer programs, is a topic of substantial scientific excitement and interdisciplinary opportunity.
The author acknowledges financial support of the Fraunhofer- Gesellschaft, ex., Germany, under grant No. 600016 (Shock Wave Induced Destruction of Tumor Cells), and grant No. 400017 (Extracorporeal, Focused Ultrasound Therapy: Effec-
tiveness, Simulation, and Planning of New Therapies). Interesting discussions with Constantin Coussios from Oxford University in the UK during my visit there, are gratefully acknowledged.