The Role of Physical Factors in Cell Differentiation, Tissue Repair and Regeneration

Physical factors may induce significant biological effects, therefore they can be applied in biomedical and biotechnological fields in order to drive and modulate biological processes. It is well known that both humoral and physical factors (in particular, but not limited to the mechanical ones) are necessary for maintaining tissue homeostasis. Both biochemical and physical factors can induce the cells to reprogram their functions to adapt dynamically to the environmental conditions.


Introduction
Physical factors may induce significant biological effects, therefore they can be applied in biomedical and biotechnological fields in order to drive and modulate biological processes.It is well known that both humoral and physical factors (in particular, but not limited to the mechanical ones) are necessary for maintaining tissue homeostasis.Both biochemical and physical factors can induce the cells to reprogram their functions to adapt dynamically to the environmental conditions.
It is evident, therefore, that the only way to approach functional tissue regeneration and repair is to supply combined humoral and physical stimuli in a dose-and time-dependent manner.For example, in vitro studies have shown that a biomimetic environment simulating pulsatile flow is an indispensable condition for the tissue engineering of functional trileaflet heart valves from human marrow stromal cells.Static controls show morphological alterations and weaker mechanical properties (Hoerstrup et al., 2002).
Studies on the role of physical factors in tissue repair and regeneration cover a very broad field that extends from investigations aimed at deepening our understanding of the physiological mechanisms of tissue repair and regeneration to biotech advances in tissue engineering, such as development of biocompatible scaffolds, 3D cell culture systems and bioreactors, which in the future must integrate the delivery of biochemical factors with the provision of physical stimuli that are equally necessary.In this chapter, far from providing a comprehensive overview of this field of studies, we introduce some issues concerning the application of physical factors in biomedicine and biotechnology and report the results of our research on the application of various physical stimuli (gravitational and mechanical stresses, laser radiation, electromagnetic fields (EMF)) for modulating cell commitment and differentiation, cell adhesion/migration, production and assembly of extracellular matrix (ECM) components, with the final aim of understanding when and how physical stimuli can be useful for promoting tissue repair and formation of functional tissue constructs.We also briefly mention how, in past centuries, the role of physical factors in biological processes has been understood and physical stimuli have been applied for therapeutic purposes.

Mechanical stresses
The importance of gravitational and mechanical factors in modulating biological processes has been known for a long time: from Galileo Galilei onwards, studies on functional adaptation of the skeleton demonstrated that bone loss or gain is related to the magnitude, direction and frequency of the stress acting upon the skeleton during application of loads (Galilei, 1632;Wolff, 1985;Rubin, 1985;Frost, 1988;Rubin, 1984;Ingber, 1998).
Within the body, cells are subject to mechanical stimulation, caused by blood circulation, ambulation, respiration, etc., which give rise to a variety of biochemical responses.It has been demonstrated that changes in inertial conditions, shear stress, stretching, etc. can strongly affect cell machinery.Cells may sense mechanical stresses through changes in the balance of forces that are transmitted across transmembrane adhesion receptors that link the cytoskeleton to the ECM and to other cells.These changes, in turn, alter the ECM mechanics, cell shape and cytoskeletal organization (Ingber, 1998(Ingber, , 1999)).A great deal of information has revealed that the ECM is a highly dynamic and elastic structure which undergoes continuous remodelling, in particular during development, angiogenesis, wound healing and other tissue repair processes.The ECM interacts with cells to provide relevant microenvironmental information, biochemically through the release of stored soluble and insoluble factors, and physically through imposition of structural and mechanical constraints (Carson, 2004).On the other hand, mechanical stimuli modulate ECM homeostasis: mechanical forces strictly regulate the production of ECM proteins indirectly, by stimulating the release of paracrine growth factors, or directly, by triggering intracellular signalling pathways leading to the activation of genes involved in ECM turnover (Chiquet, 2003).
Mechanical stimuli affect cells through poorly understood mechanotransductive pathways that lead to changes in morphology and orientation, modulation of gene expression, reorganization of cell structures and intercellular communication through both secretion of soluble factors and direct intercellular contact (Maul et al., 2011;Kang et al., 2011;Park et al., 2006;Papachroni et al., 2009;Wall & Banes, 2005;Bacabac et al., 2010;Hughes-Fulford & Boonstra, 2010).Over the past decade, in vitro studies have indicated that the transduction of physical stimuli involves the ECM-integrin-cytoskeleton network and also calcium channels, guanosine triphosphatases (GTPases), adenylate cyclase, phospholipase C (PLC) and mitogen-activated protein kinases (MAPKs), all of which play important roles in early signaling (Rubin et al., 2006;Hoberg et al., 2005;Adachi et al., 2033;Mobasheri et al., 2005;Chiquet et al., 2009;Bacabac et al., 2010;Hughes-Fulford & Boonstra, 2010).It has been demonstrated that, in endothelial cells, different genetic programs leading to growth, differentiation and apoptosis can be mechanically switched.Cells grow when they spread, die when fully retracted, and differentiate into capillary tubes if maintained at a moderate degree of extension (Chen, 1997).
The in vitro application of mechanical stretch, simulating the mechanical load to whom heart cells are exposed in vivo, initiated in adherent cultures of neonatal cardiomyocytes morphological alterations similar to those occurring during in vivo heart growth (Vandenburg, 1996).Stem cell commitment, the process by which a cell chooses its fate, and differentiation, the resulting development of lineage-specific characteristics, have been shown to be affected by cell shape (Roskelley, 1994;McBeath, 2044;Watt 1988;Spiegelman, 1983).Internal and external forces regulate cell shape and studies have shown that cell shape can control apoptosis, gene expression, and protein synthesis, in addition to stem cell fate (Chen, 1997;Thomas, 2002).
The cells belonging to tissues that resist the effects of gravity are particularly sensitive to mechanical and gravitational stimuli, which play a key role in the development and homeostasis of these tissues.Lack of gravitational and mechanical stresses leads to the formation of impaired tissues with lower mechanical properties and reduced function.
It is well established that bone adapts its mass and architecture in accordance with the external mechanical loads applied and osteocytes, terminally differentiated cells of the osteoblastic lineage, may be considered "mechanosensory cells" (Vatsa et al., 2010).They are sensitive to both stretching and fluid flow.Mechanical stimulation of osteocytes induces intercellular signaling which results in the modulation of osteoblast and osteoclast activity (Chow et al., 1998;Turner et al., 1997).Interestingly, it has been shown that the stimulation of a single osteocyte activates many surrounding cells (Vatsa et al., 2007).
Osteoblastic differentiation can be induced by applying mechanical stress, for example by stretching the surface on which the cells are attached (Cavalcanti-Adam et al., 2002).Many studies revealed that the micromotions at the interface between bone and artificial scaffolds play a key role in scaffold integration: they can promote tissue differentiation or induce bone resorption (Prendergast et al., 1997;Carter et al., 1998;Buchler et al., 2003;Stadelmann et al., 2008;Jasty et al.,1997).In a recent paper on biomechanics of scaffolds for bone tissue engineering applications, it has been stated that in the development of a scaffold it is important to take in account not only the structural integrity but also the load transmitted to the cells via the scaffold deformation (Pioletti, 2011).
A recent review of studies which investigated the importance of loading in maintaining the balance of matrix turnover in the intervertebral disk, reported about the possible role of overloading in the initiation and progression of disc degeneration and proposed a physiological/beneficial loading range as a basis on which to design loading regimes for testing tissue constructs or favouring differentiation of stem cells towards "discogenic" cells for tissue engineering (Chan et al., 2011) An overview of studies on the role of mechanical stimuli in chondrogenesis showed that uniaxial loading induces the upregulation of genes associated with a chondrogenic phenotype while multiaxial loading results in a broader pattern of chondrogenic gene upregulation, revealing that not only intensity, but also direction and other parameters which characterize the stimulation are relevant for the achievement of the final effect.The physiological multiaxial pattern of loading within articulating joints is so complex that currently, even with the most sophisticated bioreactors, it would be impossible to simulate the in vivo situation.Therefore, it has been suggested to use the body as an "in vivo bioreactor" (Grad et al., 2011).
Conditions of gravitational unloading, both real and modeled by a Random Positioning Machine (RPM), negatively affect cellular organization and ECM production in cartilage constructs, even if at different extent.(Stamenkovic et al., 2010).
Our group is conducting for several years research on the role of gravitational and mechanical stimuli in cell differentiation, tissue repair and regeneration, with particular attention to the remodelling phase.
Our studies demonstrated that gravitational unloading favours the differentiation of osteoclastic precursors (FLG 29.1 cells).After 72 hours exposure to conditions of microgravity, modelled by a RPM (angular velocity of rotation 60°/s), the cells showed a dramatic increase in apoptosis, but the viable ones showed osteoclastic-like morphology, cytoskeletal reorganization, significant changes in gene expression profile.The expression of the major osteoclastic markers Receptor Activator of Nuclear Factor Kappa-B (RANK) and Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) strongly increased and cells showed the ability to resorb bone (Fig. 1) (Monici et al., 2006).Analysing the gene expression profile of human mesenchymal stem cells (HMSC) in loading conditions (3 hours exposure to 10xg in hyperfuge), we found overexpression of genes involved in osteoblastogenesis (GLI1, NF1, MEN1) and downregulation of genes involved in adipogenesis (PPAR, FABP4) (Tab. 1) (Monici et al., 2008a).These results, in agreement with those of other authors (Kaneuji et al., 2011;Wang et al., 2010;Searby et al., 2005) reveal that mechanical/gravitational stresses induce osteoblastic differentiation while gravitational unloading and loss of mechanical stress favour adipogenesis, osteoclastogenesis and bone resorption.

Gene
In cultures of fibroblasts exposed for 3 hours to hypergravity (10xg), we observed enhanced expression of collagen I and fibronectin (20% and 30% more than control, respectively), while chondrocytes exposed to the same treatment showed a marked increase in collagen II, aggrecan and Sox 9, a transcription factor which plays a key role in chondrogenesis.Therefore, after definition of optimal range of intensity and force direction, loading can be used to stimulate ECM production by cells of the connective tissues and to favour chondrogenesis.
A series of experiments we carried out with the aim of studying the effect of gravitational unloading on processes involved in tissue remodelling demonstrated that the loss of mechanical stress causes a disregulation in laminin and fibronectin (FN) production by fibroblasts and endothelial cells (Fig. 2B) (Monici et al., 2011).In particular, FN forms a disordered and intricate network, reproducing the typical condition of fibrous scars.We hypothesized that the altered FN fibrillogenesis could be a cause of impaired ECM rebuilding and altered cell adhesion/migration and could contribute to the impairment of wound healing observed in microgravity (Midura & Androjna, 2006;Delp, 2008).Studying the behaviour of aortic endothelial cells cultured in micro-and hypergravity we found that the exposure to simulated microgravity conditions for 72 hours (angular velocity of rotation 60°/s) caused a reduction in coronary venular endothelial cell (CVEC) number.Genomic analysis revealed that proapoptotic signals increased, while antiapoptotic and proliferation/survival genes were downregulated by the absence of gravity.Activation of apoptosis was accompanied by morphological changes, with mitochondrial disassembly and organelles/cytoplasmic NAD(P)H redistribution, as evidenced by autofluorescence analysis.Moreover, cells were not able to respond to angiogenic stimuli in terms of migration and proliferation (Morbidelli et al., 2005) In contrast, after exposure to hypergravity (10xg), no significant changes were observed in cell morphology and energy metabolism.Cells remained adherent to the substrate, but integrin distribution was modified.Accordingly, the cytoskeletal network reorganized, documenting cell activation.There was a reduction in expression of genes controlling vasoconstriction and inflammation.Proapoptotic signals were downregulated.Overall, the results documented that hypergravity exposure maintained endothelial cell survival and function by activation of adaptive mechanisms.The behavior of cells derived from microcirculation was somewhat different, because the above described effects were associated with increased anaerobic metabolism and cell detachment from the substrate (Morbidelli et al., 2009).These findings demonstrate that gravitational/mechanical stress can strongly affect endothelial function and neoangiogenesis and the biological response could also depend on the different vascular districts.

Electromagnetic fields
It is said that in the first century AD an "electric fish" was used to cure headache.Paracelsus (1493-1542) studied the medical use of lodestone and, in the sixteenth century, Sir Kenelm Digby described the magnetic cure of wounds.At the end of the seventeenth century, Galvani, with his famous experiments on bioelectricity, opened the way for modern studies on physiological EMFs and the effects of external EMFs on the body.
Over the past forty years, important advances have been made in research on bioelectricity: differences in electrical potentials of plants, animals and humans have been measured (Burr, 1972), changes in voltage gradients have been correlated with morphogenetic events in plants and animals (McCaig et al., 2005), physiological currents have been found to be signals for key processes in development (Levin, 2007).
An extensive discussion on electromagnetic effects from cell biology to medicine is presented in a recent review written by Funk et al. (Funk et al., 2009), where the coupling between physical mechanisms and cell biology is discussed in depth.In a nutshell, EMFs can cause polarization of bound charges, orientation of permanent dipoles (which results in topographycal changes in molecules), drift and diffusion of conduction charges, ion bound or release from proteins, ion-channel or receptor redistribution, conformational changes of voltage-sensitive enzymes, modulation of binding kinetics, reorientation of membrane phospholipids and changes in activation kinetics of ion channels (Funk et al., 2009).
Endogenous EMFs in living tissues are generated by physiological activities, for example movements of the musculoskeletal system structures.Vibrations of human muscles induce mechanical strains and currents have been measured both during postural muscle activity (5-30 Hz) and walking (<10 Hz) (Antonsson & Mann, 1985).Muscle contractions induce in the underlying bone tissue EMFs which are important for maintaining bone mass.Bone cells are selectively sensitive to low frequencies, in particular those ranging from 15 to 30 Hz.In this narrow range of frequencies, fields as low as 0.01mV/cm affect the remodelling activity (McLeod & Rubin, 1993).It has been found that EM current densities produced by mechanical loading (e.g.1Hz during walking) in bone lie in the range 0.1-1.0mA/cm 2 (Lisi et al., 2006).Generally, physiological EMFs are characterized by extremely low frequencies (ELF), from 0 to 300 Hz, and have low intensity.
EMFs are widely used to treat musculoskeletal diseases and many studies indicated that the most effective devices use pulsed EMFs with frequencies from 1 to 100 Hz, which induce EF of the order of V/cm (Pilla, 2002).Therefore, physiological effects may be induced by EMFs characterized by low frequencies ( optimal range 8-60 Hz) and amplitudes  1 G (Funk et al., 2009).
It has been demonstrated that pulsed EMFs can increase osteoblastic differentiation and activity and, on the other hand, inhibit osteoclastogenesis, thus shifting the balance towards osteogenesis (Otter et al., 1998;Hartig et al., 2000;Chang et al., 2004).
Studies aimed at evaluating the possibility to apply EMFs to favour ligament healing and repair demonstrated that, after exposure to pulsed EMF, fibroblasts from calf anterior cruciate ligament increased migration speed and showed enhanced collagen I expression.
On the contrary, static EMF had an inhibitory effect on wound healing, which was reversed by pulsed EMF (Chao et al., 2007).
EMFs can modulate cell proliferation.The literature indicates that both intensity and frequency of the EMF are important in determining the final effect.Kwee and Raskmark (Kwee & Raskmark, 1995) have found an increase in the proliferation of human fibroblasts exposed to 0.08 mT, while Kula and Drozdz (Kula & Drozd, 1996) have shown inhibition of cell growth in murine fibroblasts exposed to 20 mT.Even trials carried out by exposing cultures of human lymphocytes have given different effects (increase, decrease or no effect in the proliferation) depending on the intensity of the applied EMF (Paile et al., 1995;Scarfì et al., 1999).
As regards frequency, many authors reported increases in proliferation of different cell types at 50 Hz frequency (Scarfì et al., 1991;Cossarizza et al., 1993).
Numerous studies have addressed the interaction between EMFs and calcium fluxes, because calcium is a principal regulator of several cellular processes.It is an activator of cyclic AMP, key molecule in triggering intracellular metabolic processes.It has been observed that the exposure to EMFs can modulate calcium concentration in a way which depends on cell type and field intensity.(Farndale, 1987;Walleczek, 1990).
The effects of EMFs on cell differentiation have been studied too.A progressive inhibition of enzyme activity and differentiation in MC-3T3 osteoblast-like cells, after exposure to 30 Hz EMF, was described by McLeod and Collazo (McLeod & Collazo, 2000).In HMSCs exposed to EMFs during chondrogenic differentiation, increase in collagen II and glycosaminoglycan (GAG)/DNA content was observed (Mayer-Wagner et al., 2010).Therefore EMFs might be a way to stimulate and maintain chondrogenesis of HMSCs and provide a new step in regenerative medicine regarding tissue engineering of cartilage.
In recent experiments aimed at studying the effects of EMFs on neuroblasts and understanding whether these effects can be useful in promoting tissue regeneration, we found that in neuroblasts (SHSY5Y human cell line derived from neuroblastoma) exposed to low frequency EMF (50 Hz; 2 mT, 3 hours) synaptophysin and TAU (microtubule-associated proteins) were overexpressed while Microtubule-Associated Protein 2 (MAP2) was downregulated.Synaptophysin participates in the formation of the channel for neurotransmitter release.TAU is associated with the protofilaments in neurites and MAP2 is a microtubule-associated protein found predominantly in the cell body.MAP2 function is not required when the cell disassembles microtubules in the cell body to give rise to the formation of neurites, while TAU is required to add new subunits to microtubules which are forming in the neurites.Moreover, in the treated neuroblasts, we observed rearrangement of microtubules and actin microfilaments, with formation of cones and cytoplasmic extensions (Fig. 3), and increase of neurofilaments, a marker of neurogenic differentiation (not yet published data).Therefore we hypothesize that EMFs can favour differentiation.The expression of synaptophysin, TAU and MAP2 returned to control values 24 h after exposure.Instead, the formation of neurites continued to progress even after 24 h, with the appearance of branched extensions.This means that the changes in protein expression are part of a complex biological response that, once triggered by exposure to the EMF, proceeds even after the cessation of the stimulus (Cerrato et al., in press) Preliminar experiments on the effect of pulsed EMF on fibroblast behaviour in wound healing models showed, in agreement with other authors (Sunkari et al., 2011), that EMF can accelerate or slow the migration of fibroblasts, depending on the properties of the applied field (data not yet published).The possibility of modulating fibroblast migration during wound healing could be very interesting: in fact it might be useful to enhance the migration of fibroblasts to promote wound healing in chronic ulcers and, in general, in cases where healing is slow, while to inhibit the migration would be beneficial to prevent the formation of fibrous scars.

Light
Ancient civilizations had learned that light can have effects on the tissues of the body: both Romans and Greeks widely used the exposure to sun for therapeutic purposes.
In ancient China, a ritual to attain immortality in use during the Tang dynasty (fifth century AD) prescribed exposure to the sun holding in the right hand a piece of green paper with the character representing the sun in red.Subsequently, the paper previously exposed to the sun should be soaked in water and eaten for "trapping" in the body the essence of the sun.At the turn of the nineteenth century and beginning of the twentieth, it was discovered the lethal effect of ultraviolet (UV) component of sunlight on microorganisms and the efficacy of red light in preventing suppuration and scarring in patients with smallpox: the basis for the modern phototherapy were laid (Barnard & Morgan, 1903;Finsen, 1901;McDonagh, 2001).
The extensive application of UV, visible, infrared (IR) radiation in biological and medical fields led to the development of suitable light sources.Actually, lasers are the latest and most advanced type of light source.
The advantages of lasers, compared to other sources, are the high intensity of radiation emitted, the directionality (which allows efficient coupling to optical fibers and focus), the monochromaticity (if needed) and, with pulsed lasers, the possibility to transfer large amounts of energy controlling the thermal effects.
Following the widespread clinical use, many studies have been conducted to investigate at the cellular and molecular level the mechanisms underlying the systemic effects produced by exposure to laser radiation.Propagation and absorption of radiation in a biological sample or in a tissue may produce photochemical, photothermal and photomechanical effects, which are able to induce a biological response (Jacques, 1992).
The actual laser systems, because of their versatility, are particularly suitable for application in biomedicine and biotechnology and the use of lasers to modulate biological and biotechnological processes has been proposed.
Hsu (Hsu et al., 2010) proved that endothelial cells pre-exposed to red-emitting laser (632 nm) and then seeded on a biomaterial surface (biomedical grade poly (carbonate) urethane) increase matrix secretion and are more resistant to flushing (greater cell retention on the graft) in comparison with non laser-exposed controls.
An overview on the state of the art in the photoengineering of bone repair showed that infrared (IR) radiation increases osteoblastic proliferation, collagen deposition and bone neoformation (Pinheiro & Gerbi, 2006) A recent review in which the photoengineering of tissue repair in skeletal and cardiac muscles is discussed, reported that exposure to lasers with red/near infrared (NIR) emission is effective in favouring muscle repair: the application of He-Ne laser irradiation significantly enhanced muscle regeneration in rats, while Ga-Al-As laser radiation reduced muscle degeneration in the ischemia/reperfusion injury in skeletal leg muscle.Photoexposure also favoured proliferation of myogenic satellite cells.In mouse, rat, dog and pig ischemic heart models, phototherapy significantly reduced (50% -70%) the formation of scar tissue after induction of myocardial infarction.Ventricular dilation was also reduced and ATP in the infarcted area increased (Oron, 2006).
In order to evaluate the effectiveness of light emitted by lasers and other sources in enhancing cell proliferation, Alghamdi and colleagues (Alghamdi et al., 2011) reviewed the literature in this specific field from 1923 to 2010.They concluded that light with wavelength ranging from 600 to 700 nm is helpful in enhancing the proliferation rate of various cell types, stem cells included.The increase in proliferation was generally associated with increased synthesis of ATP, RNA and DNA.The reviewed data indicated that the optimum value of energy density was between 0.5 and 0.4 J/cm 2 .The possibility to develop phototreatments aimed at favouring cell proliferation could be very useful in the production of vaccines and hybrid cell lines as well as in tissue engineering and regenerative medicine.
In a review concerning the literature from 1960 to 2008 on the use of laser treatments for the improvement of tissue repair, the authors stated that, despite the difficulty in comparing results obtained with different laser sources, treatment protocols and experimental models, the majority of the reviewed reports clearly indicated that laser irradiation (the most frequently used is red/NIR radiation) speeds up tissue repair (da Silva et al., 2010).
In order to evaluate the possibility to use laser treatments as a tool to stimulate cell differentiation processes and cell functions involved in tissue repair, we studied the effect of NIR-emitting lasers on HMSCs, endothelial cells and cells of connective tissues.We found that in HMSCs treated with NIR pulses emitted by a high power Nd: YAG laser (1064 nm wavelength, 200 s pulse duration, 10 Hz repetition rate, 458.65 mJ/cm2 energy fluence, 73 sec exposure) genes involved in osteoblastogenesis (GLI1, NF1, MEN1) appeared upregulated while PPAR, which is a major marker of adipogenesis, and FABP4 were downregulated, suggesting that the treatment can favour osteoblastogenesis and inhibit adipogenesis (Tab.1).Interestingly the results obtained with laser treatment are very close to those obtained by exposure of HMSCs to hypergravity (10xg) (Monici et al., 2008b).
Moreover, the Nd:YAG laser pulses resulted effective in enhancing the production of ECM molecules, such as collagen I, collagen II, aggrecan and FN in cultures of connective tissue cells.A similar increase in ECM molecules was found when the cells were exposed to hypergravity (10xg) (Monici et al., 2008b, Basile et al., 2009).
The results of experiments in which we compared the effects of gravitational loading (10xg) and laser pulses on cells belonging to tissues with antigravitational function are consistent with the hypothesis that, using suitable laser pulses, it is possible to induce transient ECM rearrangements in the cell microenvironment (cell niche) which, in turn, act as indirect "photomechanical" stimuli on the cells (Rossi et al., 2010).
Experiments carried out on fibroblasts and endothelial cells, which are responsible for ECM production and neoangiogenesis in the remodelling phase of wound healing, demonstrated that NIR pulses not only increase (30%), FN production, but also favour the ordered assembly of FN fibrils in fibrillogenesis (Fig. 2C) (Monici et al., 2011).This effect is interesting because it could improve the quality of the neoformed ECM: in fact, FN fibrils act as a template for the formation of collagen fibers and strongly affect ECM properties (Shi et al., 2010).Moreover, we observed that NIR laser pulses can also favour the formation of ordered monolayer of endothelial cells (Monici et al., 2011).This effect could be of consequence in neoangiogenesis.Finally, data obtained with preliminary experiments carried out in our laboratory show that pulsed NIR radiation enhances the production of inflammation cytochines (not yet published data).The treatment could thus have the effect of accelerating the transition from inflammatory to the remodelling phase in tissue repair.
Advanced laser systems allow to apply more complex treatment protocols, to try to potentiate or to exploit synergistically the effects produced by emissions with different characteristics.
After exposure of myoblasts to a Multiwave locked System (MLS) laser, emitting at 808 and 905 nm (continuous/interrupted and pulsed mode, respectively) we observed increased activity of enzymes involved in cellular energy metabolism and enhanced expression of MyoD (Vignali et al., 2011), an early marker of muscle differentiation.These results provide an interesting premise for the future application of Multiwave systems to favour muscle tissue repair.

Conclusion
Over the past twenty years, studies on molecular and cellular mechanisms that underlie biological responses evoked by physical stimuli have made great progress.However, in this fascinating field of study, many problems still remain and their solution will require further advances in our knowledge.
The results of our studies are a further, albeit modest, contribution to a large body of literature that shows how physical stimuli can be effective in modulating cellular functions and the production of ECM.It is obvious that the development and standardization of technologies for delivering appropriate physical stimuli, strictly controlled with regard to the intensity, frequency and timing of exposure, is a prerequisite for making in tissue engineering.

Fig. 1 .
Fig. 1.Scanning electron microscopy of a bone slice exposed to FLG 29.1 cells cultured in modelled microgravity.Adherent cells on the bone surface can be observed.Arrows indicate the sealing zone.

Fig. 2 .
Fig. 2. FN expression in CVECs (analysed by immunofluorescence microscopy): A) control, B) exposed for 72 h to modelled microgravity and C) treated with pulsed Nd:YAG laser (1064 nm).In figure B a tight network of FN fibrils appears while in figure C the FN fibrils are parallel and ordered (see arrows).

Fig. 3 .
Fig. 3. Actin expression in SHSY5Y cells (analysed by immunofluorescence microscopy): control (A) and cells exposed to EMF (50 Hz, 2 mT, 3h) analysed immediately (B) and 24h (C) after the treatment.The formation of neurites can be observed in figures B and C.

Table 1
. Gene expression profile in HMSCs.

Table 1 .
Based on the studies cited in this chapter, the table lists physical factors, treatment parameters applied, experimental models used and observed effects.